Last year, I had the delightful opportunity to appear on The Daily Show, though alas, I didn't get to meet Trevor Noah. The segment was about various groups who oppose masturbation. The most common question I get about that appearance is in relation to a statement I made disputing alleged connections between testosterone (T) and masturbation.
Some folks online claim that refraining from masturbation makes them feel more manly, more masculine, more assertive, more dominant, and more attractive to females. They allege that this effect emerges from a supposed increase in testosterone when they stopped masturbating.
These claims are often supported by reference to a very small, un-replicated study from China, which involved a very small sample of 10 males. Being interested in this study and how the analysis was conducted, colleagues have attempted to obtain the study data to verify, but have been unable to. But better research finds that testosterone isn't as simple as these claims would have it.
The role of testosterone and sexual activity, including masturbation, is a nuanced and somewhat idiosyncratic dynamic. There are many complicating factors — for instance, men's testosterone decreases when they have a baby, and cuddle the baby. Men's testosterone has a complex effect on both sexual behaviors and male relationship behaviors. In adolescent males, higher T predicts more sex and more masturbation. Higher levels of T predict more infidelity, more open relationships, in men, and lower levels of T predict longer, monogamous relationships. Men who are polyamorous appear to maintain levels of testosterone that are commensurate with the levels of T in single males.
Ultimately, the question about testosterone and sexual behavior is a question of causal direction — typically, testosterone has been assumed to be causal, and to drive behaviors. However, a rival theory suggests that testosterone levels may be influenced by behaviors, or "socially modulated." The theory that masturbatory abstinence influences testosterone levels is an interesting offshoot of this concept. A similar theory is the one that suggests that athletes shouldn't have sex, as it depletes them of competitiveness and perhaps testosterone. However, research examining sports performance and sex has found no effect whatsoever, except when the sex occurred within just a couple of hours of the sporting event.
My good friend Dr. Justin Lehmiller covered this issue here, though he didn't include a very large and significant related study, which I describe below. He does note that in females, two of three studies point to an increase in testosterone from sexual activity. Lehmiller concludes that the evidence for an effect on testosterone from abstinence is inconclusive and largely unsupported.
This study by Van Anders looked at both males and females and found that in females, higher T predicted masturbation frequency. But in males, there was no clear connection between testosterone levels and sexual desire. Men who masturbated more did have higher libidos, but this was predicted by masturbatory frequency, not testosterone levels.
This very large study from the National Social Life, Health, and Aging Project found that level of testosterone was actually increased by masturbation in older men, and that the hormone acts differently in males and females. This study involved a longitudinal analysis of a probability sample of US adults aged 57-85 and included 650 women and 620 men.
Results found that in fact, levels of testosterone, relationship quality, frequency of sex, and masturbation remained remarkably stable across the years of this study. Contrary to beliefs, these factors, including testosterone levels, really don't seem to change all that much. When the researchers looked specifically at male masturbation, they found that men level's of T increased with higher levels of masturbation, but, interestingly, found that higher levels of T didn't appear to increase masturbatory frequency. In other words, it appears to be a one-way effect. More masturbation increases testosterone, but more testosterone doesn't increase masturbation.
An important additional element of this study looked at relationship quality, hypothesizing that higher levels of testosterone predict worse relationship outcomes, a finding which past studies have supported. In males, this effect was present in this study, finding that higher levels of T predicted lower relationship quality. This finding explores the complex nuanced trade-off of testosterone — it may increase mating effort, but inhibits long-term relationships. Interestingly, more frequent sex in females appeared to actually lower testosterone in females, which the authors suggest may reflect that this sex occurs within a relationship and that lower testosterone levels improve relationship quality.
Source: PxFuel
Overall, I stand squarely behind my statement on The Daily Show that debunked claims that abstinence from masturbation increases testosterone. These claims are based on a very poor and simplistic understanding of testosterone, sexuality, and science. These guys who claim that they want higher levels of testosterone don't understand that if they did, they'd actually be more likely to have failed and unhappy relationships. They also seem to think that testosterone is a simple hormone with simple, unidirectional effects, when the science shows us that blanket statements about any hormone or neurochemical are foolishly reductive. It's actually possible that testosterone may act differently based on age, though at this point, we don't have much data to support this. However, the idea that our behaviors influence our testosterone levels does appear somewhat likely, though not apparently in the direction hypothesized by these pseudoscientific claims. Instead, it appears that sexual behaviors may increase testosterone levels in some directions, but not others, and work differently in male bodies than in females.
What are other Boron testosterone related health benefits?
Natural ways to increase Boron levels
What is the recommended Boron for testosterone supplementation and dosage?
Where can you get Boron for testosterone?
Conclusion
What is Boron?
Boron is an essential trace mineral that is believed to increase free testosterone levels, whilst blocking excess estrogen; increasing your muscle and bone strength; improving your concentration and focus; enhancing muscle coordination, decreasing inflammation and alleviating the symptoms of osteoporosis and arthritis.
How Boron and testosterone works
You may only need Boron in trace amounts; however, the impact it can have on your body is impressive as it plays a key role in maintaining homeostasis and managing your metabolism.
You see, our bodies need a healthy balance of testosterone and estrogen in order to function, and one of the easiest ways to achieve this is to ensure that your body has got plenty of Boron. Now, we're not talking about tonnes here. However, with just the right amount you can prevent these sex hormones from changing your oral microbiome balance; lowering your immunity, and causing bleeding gums.
Boron can also help your body to absorb magnesium better – which is important as together they can help assist hundreds of functions within your body – as well as impact how your body uses calcium and phosphorus.
How can Boron increase testosterone levels?
Remember how we mentioned that Boron can influence your sex hormones? It is probably most famous for its ability to boost your natural testosterone levels. In fact, studies have shown that Boron can boost your free testosterone levels by 29.5%.
In one study, where participants were given 10mg of Boron a day for 7 consecutive days (to test for plasma steroid hormones); they discovered that their free testosterone levels had risen by 28.3%.
Similarly, in a separate study on 13 participants (who'd been diagnosed with vitamin D deficiency), after giving them 6mgs of Boron for 60 days, they found that whilst Boron helped to raise their vitamin D levels, it also increased their DHEA levels by 56% and their free testosterone by 29.5%.
So how does Boron increase testosterone?
Boron basically acts as an ergogenic aid as it naturally helps to increase the amount of free testosterone available in your body (in other words, it doesn't create more testosterone but enables your existing testosterone to do its job). This is achieved by decreasing SHBG (sex hormone binding globulin).
SHBG is a protein found in your blood which is responsible for binding sex hormones (such as testosterone) and decreasing their effects.
Boron works by blocking SHBG, thus raising your free testosterone levels and enabling you to build up muscle mass and increase your strength. The key is getting the balance between Boron and testosterone right, as too much testosterone in your system will lead to more of it being bound to SHBG.
However, with the right Boron dosage, you can 'free up' your testosterone and witness a boost in your first week (and a gradual increase the remainder of the time).
Boron can also help your testosterone levels by influencing the following:
Boron and Estrogen
According to one study, Boron can block estrogen levels. When participants were given 10mg of Boron a day (over a week), they saw a decrease in estrogen of 39%.
So whilst Boron can help to raise your testosterone levels by blocking SHBG; it can also lower the presence of estrogen.
NOTE: this decrease is only temporary. If you take Boron supplement testosterone for a prolonged period (4 weeks in a row) you will see a gradual rise in estrogen over those 4 weeks. To combat this – if you're weightlifting – it is recommended that you take Boron for short 2 weeks cycles (before taking a week break) so you can maximize your results – and reap the benefits – without incurring any of the negatives.
Boron and Vitamin D
Like we mentioned earlier, Vitamin D levels are closely linked to testosterone. This is because Vitamin D acts as a testosterone booster, meaning if you are deficient in this vitamin, you'll also be low in testosterone.
Boron can help to change this by naturally supplementing your Vitamin D levels and increasing their biological half-life. In doing so, not only will this help to prolong the viability of Vitamin D within your body; its increased presence will also help to boost testosterone production, enabling you to benefit from increased muscle mass, strength, energy, and endurance – everything you need to work out.
So does Boron boost testosterone? All the evidence seems to suggest so…
What are the other Boron testosterone related health benefits?
Whilst Boron is increasing in popularity in the bodybuilding world for boron for testosterone; it was first used to treat all of the following health conditions:
Bones and joint health – Boron can help to improve your bone/joint health and alleviate the symptoms of arthritis and osteoporosis by activating osteoblasts in the bones. In one study, 50% of participants experienced improved symptoms when they took 6mg of Boron a day.
Inflammation – Boron supports many functions within the body, including reducing oxidative stress and inflammation (particularly around your joints). It helps to decrease certain inflammatory markers in your body called cytokines (in particular hs-CRP and TNF-a), which have been linked to lung and breast cancer, obesity, insulin resistance, heart disease, depression, and many more conditions.
Wound repair and healing – according to a 1990 study, using 3% boric acid on deep wounds can help to reduce healing time by up to two-thirds, as well as improve how well the wound heals. It achieves this by interacting with enzymes collagenase, alkaline phosphatase and elastase and activating fibroblasts in your skin cells and tissues.
Teeth and gum health – Boron can help to keep both of these healthy by decreasing inflammation (relating to gum disease) and assisting with bone and tissue repair. In a 2013 study, Boron was found to help encourage teeth building cells. Hormone imbalance – Boron can help both men and women by increasing your estrogen and testosterone levels.
Natural ways to increase Boron levels
You don't have to invest in Boron testosterone boosting supplements in order to benefit from this trace mineral. There are certain foods that are rich in Boron, which if you eat regularly, can help to raise your natural Boron levels.
Good sources include walnuts, almonds, Brazil nuts, beans, broccoli, chickpeas, avocados, almonds, raisins, and bananas. Simply add them to your diet, and you'll soon notice a difference.
Take for instance avocados. One whole cup of avocado can offer you 1.7mg of Boron. This means by eating just 5 avocados a day, you can raise your Boron levels and the amount of free testosterone you've got available in your body.
What is the recommended Boron for testosterone supplementation and dosage?
With no RDA set, the amount of Boron you should have each day is not entirely clear.
However, thanks to numerous scientific case studies that have been done on this mineral; research suggests it is beneficial to have around 3-10mg of Boron a day (depending on your personal goals).
Now admittedly, there is nothing stopping you from having up to 20mg of Boron a day as it is perfectly safe to have this high a dosage. However, we feel sticking to a 10mg dose is more than enough to boost your testosterone levels.
Similarly, it is important to remember that Boron is not designed for long term use. You should only use it for two-week cycles (with a one-week break in-between) to prevent it from losing its effectiveness.
Where can you get Boron for testosterone?
TestoGen is the perfect testo booster for raising your free testosterone levels.
It utilizes Boron within its formulation to help block SHBG and stop it from binding with testosterone. This ensures you have plenty of free testosterone in your system to help build up muscle mass; shift excess fat; improve muscle development, definition and recovery, and maximize your endurance and energy levels.
For more information on TestoGen visit our website.
Conclusion
Boron can offer your body a natural way to protect your testosterone levels. By blocking SHBG (which usually binds to testosterone), this can help to increase the amount of viable testosterone you've got available, meaning when you hit the gym you can hit the gym hard.
Boron can help you to achieve optimal testosterone levels for bodybuilding, enabling you to achieve the muscle mass YOU want- 100% naturally.
Our only advice is to use it in short spurts of up to two weeks max. (no more than 10mg a day) and to remember that over time its results will slow down. The most you'll see is within your first week because as your free testosterone levels begin to rise, so will the amount of SHBG in your body. This will cause it to slow down, especially if you don't take a break.
However, use Boron for only two weeks at a time and this can help to maximize your results; prevent your body from stabilizing, and give you a boost when YOU need it.
¿Qué hacer durante la Pandemia del Coronavirus si vives con diabetes?
En estos momentos que estamos viviendo, donde nuestros planes y proyectos se vienen abajo, los humanos nos damos cuenta que lo único cierto es la incertidumbre: no podemos dar nada por sentado. Debemos adaptarnos a las situaciones, ir día a día, tomar las mejores decisiones fundamentadas con la mayor información posible.
Todos hemos escuchado a los medios de comunicación haciendo hincapié que aquellas personas que viven con enfermedades crónicas son más susceptibles de contagiarse del Coronavirus y ponerse graves. La mayoría de las personas que viven con diabetes, nos han preguntado qué deben hacer para minimizar los riesgos.
Lo que realmente nos va a ayudar es llevar el mejor manejo posible de la diabetes y sus enfermedades asociadas, pero más importante aún, quedarnos en casa, para disminuir la velocidad de los contagios. El día de hoy, salir a la calle, aumenta los riesgos de que más personas se contagien al mismo tiempo, y que los sistemas de salud se saturen.
Si nos quedamos en casa, podemos aplanar la curva de contagio y tener a menos personas graves, enfermas al mismo tiempo. Eso va a ayudar a que las personas que realmente necesiten atención, sean atendidas, y a que no saturemos nuestros sistemas de salud.
Sabemos que los mejores desenlaces ocurren cuando las personas están preparadas para cualquier contingencia. A continuación, nuestras recomendaciones:
MANTENER HÁBITOS DE HIGIENE
Lavarnos las manos con agua y jabón frecuentemente, y si no tenemos, con alcohol en gel mayor al 70% (nota, para limpiar los dedos para monitoreo de glucosa capilar, es mejor lavarse con agua y jabón, o con toallitas de alcohol- algunos alcoholes en gel, pueden darnos niveles de glucosa falsamente elevados).
Evitar tocarse la cara.
Desinfectar objetos de uso cotidiano como celular y teclados de computadora.
Mantenerse hidratado.
Seguir una buena alimentación.
Mantenerse activo.
Dormir bien.
DISTANCIAMIENTO SOCIAL
Quédate en casa.
No saludar de mano, de beso, no abrazar.
Mantén una distancia de aprox. 2 metros con otras personas cuando sea posible.
Evita ir de compras en horas pico (solo compra lo necesario).
Procura hacer videoconferencias, teletrabajo, home office y clases online.
Evita lugares públicos aglomerados (centros comerciales, plazas, cines, conciertos, gimnasios, reuniones de más de 10 personas).
Sé prudente y solidario: evita visitas innecesarias a población vulnerable: mayores de 60 años, con enfermedades crónicas (diabetes, hipertensión, enfermedades cardiovasculares), inmunosupresión.
Aquellas personas que recién regresaron del extranjero, deben mantenerse en casa por 14 días de la fecha de regreso
Aquellas personas que estuvieron en contacto con algún caso sospechoso o confirmado, deben quedarse en casa, por 14 días
Si alguien tiene síntomas respiratorios, debe quedarse en casa
Si alguien tiene síntomas de Coronavirus debe ponerse en contacto con un infectólogo y seguir sus instrucciones para hacerse las pruebas pertinentes en el lugar correcto.
MANEJO ÓPTIMO DE TU DIABETES (lo que deberías hacer siempre)
Contar con tu plan de acción para días de enfermedad y tu kit completo de emergencia, con suficientes insumos (para aproximadamente un mes):
- Glucómetros
- Pilas para glucómetros, microinfusoras y otros dispositivos.
- Porta lancetas
- Lancetas
- Tiras reactivas para checar glucosa (y cetonas)
- Sensores
- Tabletas de glucosa
- Insulinas
- Medicamentos para el manejo de nuestra diabetes
- Kit de Glucagón
- Agua
- Seguir tu tratamiento, hacer tus citas de seguimiento a distancia.
- Monitorear frecuentemente la glucosa (con glucómetro o con monitoreo intermitente o continuo de glucosa)
- Mantener los niveles de glucosa la mayor parte del tiempo en rango (70-180 mg/dl) para que tu cuerpo no se entere que vive con diabetes, y tampoco el Coronavirus.
- Y si te da Coronavirus o cualquier otra enfermedad, que encuentre a tu cuerpo lo más fuerte posible, para que el desenlace sea menos grave.
SOBRE LOS SUPLEMENTOS ALIMENTICIOS
Evita auto-prescribirte.
No hay suficiente evidencia para recomendar vitaminas en general, pero a muchas personas les ayuda tomar vitamina C, vitamina D, probióticos. Si en general, te han ayudado en otras ocasiones, las puedes tomar.
Si decides tomar vitamina C, que de preferencia sea en tabletas ingeribles, no masticables. Si son masticables, que sean sin azúcar. Y saber que si tomas vitamina C y monitoreas la glucosa con sensores como el Freestyle Libre, este te puede dar valores falsamente elevados de glucosa por algunas horas posteriores a la ingesta de la vitamina C, por lo que antes de tomar decisiones de corrección con insulina, debes confirmar tu glucosa con glucometría capilar (piquete de dedo con porta lancetas y lancetas).
Tal vez el Coronavirus, al recordarnos nuestra vulnerabilidad, y poner una pausa en nuestra acelerada vida, nos está recordando la importancia de vivir plenamente cada momento.
En estos momentos, donde nuestros planes cambian, y nos reconocemos mortales, debemos tener la capacidad de elegir ¿a qué nos arriesgamos? y ¿a qué no? ¿Qué riesgos realmente valen la pena en mi vida? y ¿cuáles no? El día de hoy, la elección correcta es quedarnos en casa.
CITAS A DISTANCIA
Clínica EnDi pone a su disposición, mantener todas sus citas ya programadas a distancia, por favor descarga datos de tus dispositivos de monitoreo de glucosa, microinfusora antes de tu sesión.
Vitamin E is a compound that plays many important roles in your body and provides multiple health benefits. In order to maintain healthy levels of vitamin E, you need to ingest it through food or consume it as an oral supplement. Read on to find out which foods are recommended sources of this essential nutrient, along with other basics to know about vitamin E.
Vitamin E is classified as an antioxidant. This means that vitamin E helps to destroy harmful compounds called free radicals that can build up in your body. Free radicals cause damage to cells through oxidative stress, and they've been linked to aging and health problems such as cancer and heart disease. Getting enough vitamin E on a daily basis may help to combat these conditions by protecting the outer membranes of your cells from free radical damage.
Photo Courtesy: Leren Lu/Photodisc/Getty Images
Vitamin E also plays an important role in your immune system and your body's ability to fight infection. At certain doses, vitamin E has been shown to stimulate the function of T cells — a type of cell that responds to pathogens that cause disease. Recent studies have shown that increasing your vitamin E intake may correlate with a stronger immune response and greater resistance to infection.
Vitamin E is present in the following whole foods:
As people age, their daily dosage recommendation for vitamin E increases. For these standard recommended daily doses, a healthy balanced diet is usually sufficient for getting the required amount of vitamin E:
Photo Courtesy: skynesher/E+/Getty Images
Birth to 6 months: 4 milligrams (mg)/day
1 to 12 months: 5 mg/day
1 to 3 years: 6mg/day
4 to 8 years: 7mg/day
9 to 13 years: 11mg/day
14+ years, including adults and seniors: 15mg/day
A person's age, gender and health conditions can help determine their recommended daily dose of vitamin E. Always consult a healthcare professional before altering your intake of vitamin E from the recommended daily dosage.
Taking Vitamin E Supplements
If it becomes necessary, such as if you become deficient in vitamin E, your doctor may advise you to start taking vitamin E supplements. Vitamin E supplements may come with some minor side effects if you take more than the recommended daily amount or if you combine these supplements with certain medications.
Photo Courtesy: Moyo Studio/E+/Getty Images
For example, vitamin E may increase the risk of bleeding if you take it with anticoagulants (also called blood thinners) like warfarin. Other medications, like chemotherapy drugs and cholesterol-lowering drugs, have potential harmful interactions when combined with vitamin E supplements. Talk to a healthcare professional before introducing daily vitamin E supplements, and be sure to discuss current medications you're taking.
Signs of a Vitamin E Deficiency
Because most people are able to get a sufficient amount of vitamin E through their normal daily diet, vitamin E deficiency is rare and typically related to an underlying health issue. For example, because vitamin E is a fat-soluble nutrient, there's a risk for vitamin E deficiency in people whose bodies are unable to absorb fat properly. Premature infants may also become deficient in vitamin E.
Photo Courtesy: LaylaBird/E+/Getty Images
When vitamin E deficiency does occur, these are some of its common symptoms:
Muscle weakness
Unsteady gait
Nerve pain or numbness
Impaired vision
Vitamin E deficiency can be detected with a blood test. It typically resolves with minor changes in your diet or the addition of vitamin E supplements. It's important to address any out-of-range vitamin E levels with your doctor. Chronic deficiencies may prevent your immune system from functioning normally.
Why you shouldn't take vitamins and supplements past their expiration date
This article was medically reviewed by Kailey Proctor, MPH, RDN, CSO, a board-certified oncology dietitian at the Leonard Clinical Cancer Institute with Mission Hospital.
Vitamins and supplements do expire even if you don't see an expiration date on the package. d3sign/Getty Images
Expired vitamins or supplements are not toxic, but they are less potent and effective.
Taking less-potent vitamins can be dangerous if you need a certain dosage to treat a condition.
To prevent vitamins from expiring, store them in a cool, dark, and dry environment.
Visit Insider's Health Reference library for more advice.
The use of supplements has increased in recent years. Designer herbs, blends, and formulations of vitamins are endorsed by popular celebrities and packaged to be pleasing. But do we know how they should be stored to keep from degrading or expiring? Here is some information for you to keep your vitamins potent and safe.
Yes, vitamins do expire
The FDA doesn't require an expiration date which means most vitamins do not have one listed on the package, but they do, in fact, expire. However, it's still safe to take vitamins after their best by date.
"Vitamins are still fine to take after [their expiration], but they may lose their potency or begin to break down," says Chelsea Tersavich, PA-C and Nutrition Outreach Fellowship participant for the Physician Assistant Foundation. "The breakdown can be to the vitamin itself or the [non-vitamin] compounds it is mixed with to create the vitamin tablet, pill, chewable, or gummy."
There is no consensus on when vitamins may expire or begin to lose their potency as every vitamin will expire at a different rate. That's because each vitamin formulation is unique, containing different ingredients, amounts, and packaging.
However, tablets and pills last longer than liquids or gummy vitamins because their hard exterior resists moisture. They are also able to stand larger fluctuations in temperature because they are protected.
"Chewable and gummy vitamins have fillers that although make them more enjoyable to take, make them more susceptible to absorbing moisture and therefore breaking down faster than tablets and pills. Additionally, gummy vitamins have a much smaller temperature range they can be exposed to without melting," Tersavich says.
How to store your vitamins properly
It's important to store any medications away from environmental factors like sunlight, humidity, water, and even air because these will reduce the vitamin's potency or break down their composition.
Here is a list of common vitamins and what elements impact them:
Riboflavin (B2): Sunlight and oxygen
Folic acid (B9): Sunlight and oxygen
Biotin: Sunlight and oxygen
Vitamin C: Humidity
Vitamin D: Humidity
Here are some tips to properly store your vitamins:
Keep at room temperature, between 46 and 77 degrees Fahrenheit.
Avoid storing them in the refrigerator, which has high moisture levels.
Find a dark location, to protect from sunlight.
Don't store them in the bathroom as humidity from the shower can damage vitamins.
Keep them in the original packaging.
Avoid storing in the kitchen if cabinets are glass and get direct sunlight
Additionally, look for a storage spot that is convenient to your personal routine. For example, You wouldn't want to store vitamins high up on an out-of-sight shelf as you'll be more likely to forget them. Instead, place them by the coffee machine or on your nightstand, where you'll see them first thing in the morning or before you go to bed.
Insider's takeaway
While vitamins don't truly degrade, time, temperature and humidity can take a toll on their effectiveness. It's very important to store them properly to preserve their potency and effectiveness. Even though vitamins are all different in how they break down, the best way to store them needs to be away from all damaging environmental factors like water, sun, and air.
Jessica Farthing is a freelance writer lucky enough to live on the coast of Georgia. In addition to exploring topics for Insider, she's written for Eating Well Magazine, Eat This, Not That, MSN, YourTango, and many other publications discussing food, lifestyle, health, and disability.She enjoys exploring health topics and sending the links to her three children, trying to convince them to take care of themselves. Life as an empty nester is challenging her to take on those unfinished projects, like a cookbook and a thriller novel or two. Jessica spends her downtime riding her horses, Henry and Limerick, and working off those sore equestrian muscles on her yoga mat. She and her husband Paul are enjoying their dinners for two. You can follow her on Instagram at @saltairsavannah.
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More: Health Explainers Health Vitamins Supplements
For the biologic machinery of the body to work correctly, vitamin C is required. Most animals can manufacture vitamin C in their bodies and do not require vitamin C in the diet. Guinea pigs, humans, and other primates share a gene mutation that makes production of vitamin C impossible. For this reason, these animals require a dietary source of vitamin C.
Guinea pigs who do not receive enough vitamin C in their diet can suffer from vitamin C deficiency (commonly known as scurvy in humans). Affected guinea pigs may have a rough hair coat, lack of appetite, dental pain, delayed wound healing, lameness, and an inability to fend off infections. Guinea pigs with a slight vitamin C deficiency may show no visible signs of disease; however, their immune system may be compromised leading to decreased ability to fight off other illnesses
The amount of vitamin C required by adult guinea pigs is about 20-25 mg/day and up to 30-40 mg/ day for pregnant guinea pigs. Guinea pig pellets are fortified with vitamin C. However, because vitamin C is a water-soluble vitamin it loses its potency over time and guinea pig pellets usually have a shelf life of 90 days after the manufacturing date. After this time, the value of the vitamin C can diminish greatly. Many pet guinea pigs have been diagnosed with a vitamin C deficiency even though they were on a good quality guinea pig pellet. Timothy and other hays should be the foundation of any guinea pig diet. Herbivores require this source of food for good digestion, good movement of food through their system and for dental health maintenance. Unfortunately, the vitamin C content of grasses, grass hays, and legume hays like alfalfa is very low. So how do we get this important nutrient into our pets?
Supplements
Some sources still recommend putting vitamin C in the drinking water either by grinding up tablets and dissolving them or by using vitamin C syrup. However, this is not the most effective way of supplementation due to several problems. The most serious problem is that vitamin C supplements in the water change its taste, and the guinea pig may not drink enough water to get adequate amounts. When this happens, not only do guinea pigs not get enough vitamin C, but they may not drink enough water leading to mild to moderate dehydration. Chronic dehydration may lead to other medical problems such as urinary tract disease. Another issue with vitamin C supplementation of the water involves the stability of the vitamin. Vitamin C is degraded quickly in light, heat, and moisture. Most water bottles are clear so they let in light. It is estimated that after about 8 hours, the amount of active vitamin C in the water is only 20% of the original amount added. For these reasons, adding vitamin C to guinea pig's drinking water is not recommended.
The best way to supplement vitamin C is either through vitamin C tablets or liquids given directly to the guinea pig or through the fresh foods they eat. Abbott and Proctor and Gamble make flavored vitamin C liquids for children. Abbott's product (Cecon) is considered by the company to be stable for 3 years from the date of manufacture (1). Proctor and Gamble states that Vicks C drops is stable for 2 years from date of manufacture (1). Other companies have flavored tablets that are listed on the label as being good for about 2 years from date of manufacture (e.g. Kirkman Labs). Oxbow company makes a vitamin C tablet (GTN-50C) for guinea pigs. These tablets are flavored and accepted by many pets as treats. Each tablet contains 50 mg of encapsulated vitamin C. Oxbow states the vitamin C is stable for 1 year from the date of manufacture1. Always be sure to check the expiration date of the product used and do not use past that date.
When using preparations of vitamin C rather than fresh foods, it is important to be sure your pet accepts the treats. Experimenting with different flavors will help you see what your guinea pig enjoys. The tablets can be given by hand if accepted or crushed and sprinkled on a special green (moistened) they like. The liquids can either be given by dropper or syringe (if accepted easily). If you have to fight to get your pet to take the liquid, try something else. Try different methods of feeding different supplements and see what works for you. A trial of a week for each method will give you a good idea what will work and what will not.
Fresh Foods Rich in Vitamin C
Probably the best and perhaps the healthiest way for your guinea pig to get a proper amount of vitamin C per day is through feeding fresh foods that are rich in vitamin C. The foods mentioned below are not only rich in vitamin C but also in a variety of other vitamins and trace minerals. In addition, chewing on these foods is good for the teeth and allows for variety in their diet. A varied diet can be mentally stimulating and is actually an environmental enrichment.
Many foods contain vitamin C. The foods that contain the highest levels of vitamin C per weight of food item are considered vitamin C-rich. However, to supplement a guinea pig with vitamin C, we must also consider the acceptability of the food item to the pig. Unlike children, we cannot make them stay at the dinner table until they have cleaned their plate. We also have to consider whether the vitamin C rich food item is appropriate for an herbivore like the guinea pig.
Guinea pig bath in the sink
Photo courtesy of Depositphotos
There are many Internet sites that report nutritional analysis of food items. Unfortunately, the vitamin C content reported varies between sites. This may be related to misinformation or to a failure to report how the analysis was done, the weight of the food item tested, the way the food was prepared (e.g. cooked, raw), and/or the part of the plant that was tested (e.g., leaves, flowers, stalks). For the purposes of this article, the authors depended on food analysis at two sites. The first site consulted was the USDA's report on vitamin C content. For food items not analyzed raw by the USDA, we consulted a web site called Nutrition Data. This web site correlated well with the USDA site on many food analyses and was deemed reliable.
Below you will find a table listing what the authors consider to be excellent, good, fair, and poor choices for vitamin C supplementation in guinea pigs. In this chart, you will find the food item (first column) and the amount of that item needed to provide your pet with 25 mg vitamin C per day (fourth column).
The authors debated about including the items considered a poor source of vitamin C in this article. However, many owners feed these items as treats. For this reason, we opted to make mention of some very poor choices of vitamin C sources. Since we started this list by perusing the USDA report for foods with no less than 30mg vitamin C per measure, if you do not see a food on this list, it is likely to be a very poor source of vitamin C. However, if there is any doubt, consult the web sites above and search for your food item. All foods mentioned are raw unless otherwise noted.
Please do not forget to feed your guinea pig hay, dark leafy greens and vitamin C. A balanced diet rich in vitamin C is the best thing you can provide for your pet.
The USDA provides a large nutrient database for vitamin C.
Enjoy feeding vitamin C and enriching the diet and life of your pig!
(1) These figures regarding stability were obtained through contact with each company's technical service department.
EXCELLENT Choice for Supplementing Guinea Pig Diet
Vitamin C (L-ascorbic acid, ascorbate, VC) is a potential chemotherapeutic agent for cancer patients. However, the anti-tumor effects of pharmacologic VC on hepatocellular carcinoma (HCC) and liver cancer stem cells (CSCs) remain to be fully elucidated. Panels of human HCC cell lines as well as HCC patient-derived xenograft (PDX) models were employed to investigate the anti-tumor effects of pharmacologic VC. The use of VC and the risk of HCC recurrence were examined retrospectively in 613 HCC patients who received curative liver resection as their initial treatment. In vitro and in vivo experiments further demonstrated that clinically achievable concentrations of VC induced cell death in liver cancer cells and the response to VC was correlated with sodium-dependent vitamin C transporter 2 (SVCT-2) expressions. Mechanistically, VC uptake via SVCT-2 increased intracellular ROS, and subsequently caused DNA damage and ATP depletion, leading to cell cycle arrest and apoptosis. Most importantly, SVCT-2 was highly expressed in liver CSCs, which promoted their self-renewal and rendered them more sensitive to VC. In HCC cell lines xenograft models, as well as in PDX models, VC dramatically impaired tumor growth and eradicated liver CSCs. Finally, retrospective cohort study showed that intravenous VC use was linked to improved disease-free survival (DFS) in HCC patients (adjusted HR = 0.622, 95% CI 0.487 to 0.795, p < 0.001). Our data highlight that pharmacologic VC can effectively kill liver cancer cells and preferentially eradicate liver CSCs, which provide further evidence supporting VC as a novel therapeutic strategy for HCC treatment.
Introduction
Liver cancer is the sixth most frequent cancer and the second leading cause of cancer-related death worldwide.1 Hepatocellular carcinoma (HCC) accounts for over 80% of primary liver cancer cases and it is characterized by a high recurrence rate and heterogeneity.2 These pathological properties may flow from cancer stem cells (CSCs), which are capable of self-renewal and differentiation responsible for tumor progression, metastasis, and chemotherapy-resistance.3,4 Therefore, eradication of CSCs is emerging as a novel treatment strategy for liver cancer.
Vitamin C (L-ascorbic acid, ascorbate, VC), an important natural antioxidant, has a controversial history in cancer treatment. In the 1970s, Pauling and Cameron performed clinical trials showing efficacy of intravenous ascorbate in prolonging the survival of patients with terminal cancer.5,6,7 However, these researches were heavily criticized after subsequent double-blind and placebo-controlled trials using oral VC at the Mayo Clinic failing to show any benefit.8,9 It was recognized later that the route of VC administration was the key reason for the discrepancy. The originally reported studies using intravenous VC produces much higher plasma concentrations than the subsequent trials employing oral VC.10 More recently, Chen et al. have revealed that ascorbate at pharmacologic concentrations (0.3–20 mM) achieved only by intravenously (i.v.) administration selectively kills a variety of cancer cell lines in vitro, but has little cytotoxic effect on normal cells.11,12,13 Furthermore, high-dose parenteral VC administration represses the growth of numerous cancers in xenografts models including pancreatic cancer, ovarian cancer, prostate cancer, colon cancer, mesothelioma, breast cancer, and neuroblastoma.13,14,15,16 These observations have reactivated interest in anti-tumor effect of pharmacological VC globally. Yet, the detailed mechanisms underlying VC-induced cytotoxicity and the potential mechanisms modulating the differences in the sensitivity of cancer cells to VC are poorly understood. Additionally, whether VC has toxic effect on CSCs remains an open question.
Ascorbic acid (the reduced form of vitamin C) is specifically transported into cells by sodium-dependent vitamin C transporters (SVCTs).17 Two different isoforms of SVCTs, SVCT-1 (encoded by the SLC23A1 gene) and SVCT-2 (SLC23A2), have been cloned.18 SVCT-1 is predominantly expressed in epithelial tissues, whereas the expression of SVCT-2 is ubiquitous.19 With respect to liver, SVCT-2 is the key protein responsible for VC uptake.20 SVCT-2 has higher affinity for VC than SVCT-1.21 Furthermore, genetic variations in SVCT-2 are closely associated with the risk of various cancers including gastric cancer, lymphoma, and head and neck squamous cell carcinomas.22,23,24 However, SVCT-2 expression and function in cancer and CSCs remain poorly characterized. So we hypothesize that SVCT-2 expression mainly responsible for VC uptake is linked to the differential susceptibility of liver cancer cells and CSCs to VC-induced cytotoxicity. Moreover, we investigate the mechanisms underlying VC-induced cell death and expression levels of SVCT-2 in HCC and CSCs.
Results
SVCT-2 is highly expressed in liver CSCs and is required for the maintenance of liver CSCs
As illustrated in Fig. 1a, SVCT-2 was highly expressed in HCC samples in comparison to peri-tumor tissues. Furthermore, we employed tissue microarray immunohistochemistry to examine the prognostic significance of SVCT-2 expression in clinical tumor samples from cohorts of HCC patients (n = 104) (Fig. 1b). Importantly, high expression (grade 2+/3+) of SVCT-2 was in agreement with poorer overall survival (OS) of HCC patients (Fig. 1c) and more aggressive tumor behavior (Supplementary Table 1) compared to low or grade 0/1+ SVCT-2 expression. Intriguingly, SVCT-2 expression was positively correlated with stemness-related genes Sox-2, Oct-4, Lin28 or CSC marker CD133 (Fig. 1d, e). Sphere formation is well established to enrich CSCs on the basis of their self-renewing capacity.3 In vitro, we found that SVCT-2 expression was dramatically increased in the spheres derived from HCC cells compared with the corresponding adherent cells (Fig. 1f, g). Then, we isolated CD133+25 or OV6+26,27cell populations from both cultured HCC cell lines and HCC patient samples. Elevated expression of SVCT-2 was also detected in CD133+ or OV6+ cell populations than CD133− or OV6− cell subsets (Fig. 1h, i), suggesting that SVCT-2 is enriched in liver CSCs. To further determine the pathological role of SVCT-2 in liver CSCs, we knocked down SVCT-2 in Huh7 cells. SVCT-2 silencing dramatically declined expressions of stemness-related markers at both mRNA and protein levels (Fig. 2a, b). Additionally, sphere formation was markedly decreased in shSVCT-2 cells compared to shCtrl cells (Fig. 2c). Furthermore, knockdown of SVCT-2 significantly reduced the proportion of CD133+ or EpCAM+ cells (Fig. 2d) as well as the resistance to chemotherapeutic drugs in both parental and cisplatin-resistant or sorafenib-resistant Huh7 cells, which were established by continuous stepwise selection in increasing concentration of cisplatin or sorafenib from the parental cell lines over several months in our lab (Fig. 2e). In in vivo models, SVCT-2 deficiency remarkably decreased xenograft tumor growths and weights (Fig. 2f, g). Consistent with in vitro results, the expressions of stemness markers (CD133 and Oct-4) were reduced in shSVCT-2 cells-derived tumor tissues compared to shCtrl cells-derived tumor tissues from mice (Fig. 2h, i). Moreover, SVCT-2 deficiency promoted apoptotic markers (cleaved caspase 3 and cleaved poly(adenosine diphosphate–ribose) polymerase (PARP)) expressions in vivo (Fig. 2h, i). Altogether, these data suggest that SVCT-2 is preferentially expressed in liver CSCs and is required for the maintenance of liver CSCs.
Fig. 1
SVCT-2 is highly expressed in liver CSCs. a SVCT-2 expression was verified in HCC patient samples by immunoblotting. Samples derived from the same experiment and gels/blots were processed in parallel. b SVCT-2 immunohistochemistry staining in HCC tumor microarray (n = 104). Staining intensity grade was indicated in the upper right corner. Low SVCT-2 expression: grade 0/1+; high SVCT-2 expression: grade 2+/3+. Scale bars = 100 μm. c Kaplan–Meier analysis of overall survival in 104 HCC patients according to SVCT-2 expression. d SVCT-2 and Sox-2 expressions were detected by quantitative RT-PCR, followed by correlation analysis in HCC tissues. e Left: correlation analysis of SVCT-2 expressions with Oct-4 or CD133 expressions in HCC tissues. Right: IHC analysis of SVCT-2, Oct-4, and CD133 expressions in HCC tissues. Scale bars = 100 μm. f, g SVCT-2 is preferentially expressed in tumorspheres generated from HCC cells than nonspheres by qPCR (f) and immunoblotting (g). Samples derived from the same experiment and gels/blots were processed in parallel. h, i Relative expression of SVCT-2 was detected in CD133+ or OV6+ cell populations enriched from HCC cells (h) and HCC samples (i) in comparison to those of CD133− or OV6− cell subsets. P peri-tumor, T tumor
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Fig. 2
SVCT-2 is required for the maintenance of liver CSCs. a qRT-PCR analysis for stemness markers in shSVCT-2 cells and shCtrl cells. b Western blot analysis showing SVCT-2, CD133, and Oct-4 expressions in shSVCT-2 cells and shCtrl cells. Samples derived from the same experiment and gels/blots were processed in parallel. c shSVCT-2 cells and shCtrl cells were cultured for sphere-formation assays. Scale bars = 150 μm. d Flow cytometric analysis for the proportion of CD133+ or EpCAM+ cells in shSVCT-2 cells and shCtrl cells. e Left: shSVCT-2 and shCtrl parental Huh7 cells were treated with indicated concentrations of cisplatin and sorafenib for 48 h. Right: shSVCT-2 and shCtrl cisplatin-resistant or sorafenib-resistant Huh7 cells were treated with indicated concentrations of cisplatin and sorafenib for 48 h. Cell viability was determined by the CCK-8 assay. f, g shSVCT-2 and shCtrl cells (1 × 106) were injected subcutaneously into nude mice. Tumor sizes were measured every week (f). After ~21 days of treatment, mice were euthanized and total tumor weights were measured (g). h Western blot analysis showing SVCT-2, CD133, Oct-4, and cleaved caspase 3 expressions in shSVCT-2 cells and shCtrl cells-derived tumor tissues from mice. Samples derived from the same experiment and gels/blots were processed in parallel. i IHC analysis showing SVCT-2, CD133, Oct-4, cleaved PARP, and cleaved caspase 3 expressions in shSVCT-2 cells and shCtrl cells-derived tumor tissues. Scale bars = 100 μm. Data are representative of at least three independent experiments and shown as mean ± s.d. (*p < 0.05; **p < 0.01; ***p < 0.001)
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SVCT-2 determines the differential susceptibility to pharmacological VC-induced cell death
As evidenced by clinical pharmacokinetics analyses,10 pharmacologic concentrations of plasma VC higher than 0.3 mM are achievable only from i.v. administration. To mimic potential clinical i.v. use, we treated five human HCC cell lines and two immortalized liver cell lines (HL-7702 and QSG-7701) with VC concentrations ranging from 0.3 to 1.5 mM. The viabilities of HCC cells were dramatically decreased after exposure to VC in dose-dependent manner, whereas the cytotoxicity of VC to immortalized liver cells was much weaker (Supplementary Fig. 1a, b). For all HCC cell lines, VC concentrations leading to 50% decrease in cell survival (IC50 values) were less than 1 mM, whereas IC50 values of VC in immortalized liver cell lines were obviously higher than 1 mM (Fig. 3a). These tested cells could be divided into three groups according to IC50 value of VC, the immortalized liver cells (HL-7702 and QSG-7701) with IC50 > 1 mM, VC-resistant HCC cells (SMMC-7721 and HCC-LM3) with 0.7 mM < IC50 < 1 mM, and VC-sensitive cells (Huh7, CSQT-2, and PLC/PRF/5) with IC50 < 0.7 mM (Fig. 3a). The inhibitory effect of VC was further confirmed in HCC-LM3 and Huh7 cell xenograft models in vivo. As shown in Fig. 3b, tumor derived from VC-sensitive Huh7 cells exhibited lower relative tumor weight compared with VC-resistant HCC-LM3 cells after VC treatment, in consistent with in vitro findings.
Fig. 3
SVCT-2 determines the differential susceptibility to pharmacological VC-induced cell death. a IC50 values of VC in HCC cell lines and immortalized liver cell lines. These cells were treated with various concentrations of VC for 48 h. Cell viability was determined by the CCK-8 assay. b Relative weights of tumors from HCC-LM3 cells and Huh7 cells subcutaneously inoculated into nude mice after VC or PBS treatment. c SVCT-2 mRNA expressions in HCC cell lines and immortalized liver cell lines were detected by qRT-PCR. d Correlation between SVCT-2 mRNA expressions and IC50 values of VC in HCC cell lines and immortalized liver cell lines. e Western blot analysis showing expressions of SVCT-2 in HCC cell lines and relative normal liver cells. Actin served as a loading control. Samples derived from the same experiment and gels/blots were processed in parallel. f Correlation between SVCT-2 protein expression and IC50 values of VC in HCC cell lines and relative normal liver cells. g Intracellular VC concentration in the tested cells after exposure to 2 mM VC for 1 h. h Correlation between SVCT-2 mRNA expression and intracellular VC concentration in tested cells after VC treatment. i Left: Huh7 cells were transfected with SVCT-2-shRNA or scramble shRNA and the SVCT-2 expression was analyzed by immunoblotting. Samples derived from the same experiment and gels/blots were processed in parallel. Right: Huh7 cells transfected with SVCT-2-shRNA or scramble shRNA were treated with indicated doses of VC for 48 h. Cell viability was determined by the CCK-8 assay. j Intracellular VC concentrations in shSVCT-2 cells and shCtrl cells after treatment with VC at the indicated doses for 1 h. Data are representative of at least three independent experiments and shown as mean ± s.d. (*p < 0.05; **p < 0.01; ***p < 0.001)
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To investigate whether the difference in susceptibility to VC results from distinct concentrations of VC flow into cells, we initially examined the expressions of SVCT-1 and SVCT-2, both of which are responsible for VC uptake into cells, in tested cells. Interestingly, both the mRNA and protein levels of SVCT-2 were inversely correlated with IC50 values of VC in tested cells (Fig. 3c–f), whereas expressions of SVCT-1, which has lower affinity for VC than SVCT-2,21 were irrelevant to the IC50 values (Supplementary Fig. 1c, d). Moreover, SVCT-2 expression levels were positively correlated with intracellular VC concentrations in tested cells after VC treatment (Fig. 3g, h). To further explore the role of SVCT-2 in VC sensitivity, we knocked down SVCT-2 expression via short hair RNA (shRNA) on Huh7 cell line expressing high levels of SVCT-2 (Fig. 3i). Compared with control cells, the viabilities of shSVCT-2 cells significantly increased following VC treatment, implying resistance to VC (Fig. 3i and Supplementary Fig. 1e, f). Meanwhile, VC flow into shSVCT-2 cells dramatically decreased (Fig. 3j). These results indicate that differential sensitivity to VC may result from variations in VC flow into cells, which is dependent on SVCT-2 expression.
Pharmacological VC preferentially kills liver CSCs in vitro
In light of above findings showing enrichment of SVCT-2 in liver CSCs, we next evaluated whether liver CSCs were more sensitive to VC-induced cell death. Intriguingly, in contrast to the effect of conventional chemotherapeutic agent cisplatin, to which CSCs are known to resist,28 VC treatment markedly downregulated the expressions of stemness-related genes and reduced the percentage of CD133+,25 EpCAM+,29 or OV6+26,27 CSCs both in HCC cells and tumorspheres (Fig. 4a–c, h). We further determined the effect of pharmacologic VC on liver CSCs self-renewal, as evidenced by the capacity of CSCs to form spheroids in vitro. As a result, high-dose VC significantly impaired both the tumorspheres initiation (Fig. 4d, e) and the growth of established tumorspheres derived from HCC cells (Fig. 4f, g) in a time-dependent and dose-dependent manner.
Fig. 4
Pharmacological VC preferentially eradicates liver CSCs in vitro. a qRT-PCR analysis for stemness markers in the HCC cells untreated or treated with 0.5 mM VC or 0.5 μg/ml cisplatin for 48 h. b qRT-PCR analysis for stemness markers in tumorspheres derived from HCC cells untreated or treated with 0.5 mM VC for 48 h. c Flow cytometric analysis for the proportion of CD133+ or EpCAM+ cells in HCC cells untreated or treated with 0.5 mM VC or 0.5 μg/ml cisplatin for 48 h. d, e Representative images of the HCC cells cultured under non-adherent condition with VC at 0.3–1 mM or PBS (control) for 5 days (d). Quantification of tumorspheres in the same experiment (e). Scale bars = 150 μm. f, g Representative images of tumorspheres at day 5 of culture treated with the indicated concentrations of VC (f). Number of tumorspheres was counted every 5 days for 10 days (g). Scale bars = 150 μm. h OV6+ and OV6− cells obtained by magnetic sorting from Huh7 cells were treated with 0.5 mM VC or 0.5 μg/ml cisplatin for 48 h. Data are representative of at least three independent experiments and shown as mean ± s.d. (*p < 0.05; **p < 0.01; ***p < 0.001)
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SVCT-2-dependent mechanisms of pharmacological VC-induced cell death
Intracellular reactive oxygen species (ROS) levels increased in two HCC cells differentially expressing SVCT-2 protein after exposure to VC. More ROS was detected in Huh7 cells with relative higher SVCT-2 expression compared to HCC-LM3 cells (Fig. 5a). The antioxidant, N-acetyl-L-cysteine (NAC), preventing VC-induced ROS production (a ROS scavenger), completely restored the viability and colony formation among VC-treated cells (Fig. 5b and Supplementary Fig. 2a). Furthermore, DNA double-strand damage was found following VC treatment, as shown by phosphorylation of histone 2AX (H2AX) depending on VC concentration. DNA damage was prevented by NAC and H2O2 (a major form of ROS) induced similar effects (Fig. 5c). Additionally, SVCT-2 knockdown markedly reduced expression of phosphorylated H2AX (p-H2AX) induced by VC, suggesting VC-induced DNA damage is dependent on SVCT-2 (Fig. 5d). A PARP inhibitor, Olaparib, inhibiting DNA repair and enhancing DNA damage, significantly increased VC-induced cell death (Supplementary Fig. 2b, c). Addition of cisplatin, a conventional chemotherapeutic regimen, to VC enhanced DNA damage (Supplementary Fig. 2d) and exhibited an synergistic effect on cell death in comparison to either drug alone, as evidenced by combination index (CI), which was calculated with isobologram principles30 to determine synergism (CI < 1), additive effect (CI = 1), or antagonism (CI > 1) (Supplementary Fig. 2d).
Fig. 5
VC uptake via SVCT-2 increases intracellular ROS and subsequently causes DNA damage and ATP depletion, further leading to cell cycle arrest and apoptosis. a Quantification of ROS levels in HCC cells. Cells were treated with 2 mM VC for 1 h after pretreatment with 2 mM NAC. Then, the cells were incubated with DCF-DA for 30 min and analyzed by flow cytometer. b HCC cells were treated with 1 mM VC for 48 h after pretreatment with 2 mM NAC. Cell viability was determined by the CCK-8 assay. c Western blot analysis showing expressions of p-H2AX in HCC cells exposed to 0.5 mM VC with or without NAC for 48 h and p-H2AX induced by VC or H2O2 in a dose-dependent manner. Samples derived from the same experiment and gels/blots were processed in parallel. d Western blot analysis showing p-H2AX in shSVCT-2 cells and shCtrl cells treated with VC for 48 h. Samples derived from the same experiment and gels/blots were processed in parallel. e ATP levels of HCC cells at different time points after treating with 2 mM VC. At 3 h, addition of NAC before VC reversed the declines of ATP levels induced by VC treatment in HCC-LM3 cells (purple square) and Huh7 cells (green triangle). f ATP levels of shSVCT-2 cells and shCtrl cells at different time points after VC treatment. ATP was normalized to the total cellular protein in each sample. g Flow cytometric quantification of cell cycle phase of HCC cells treated with VC or H2O2 for 48 h. h Flow cytometric quantification o Annexin V-FITC/PI double-staining of HCC cells treated with VC or H2O2 for 48 h. Early apoptosis: Annexin V positive and PI negative; late apoptosis: both Annexin V and PI positive. i HCC cells were treated with 1 mM VC for 48 h after pretreatment with the pan-caspase inhibitor Z-VAD-FMK for 1 h. Cell viability was determined by the CCK-8 assay. j Western blot analysis showing SVCT-2, p-H2AX, p21, and cleaved PARP expression in shSVCT-2 tumorspheres and shCtrl tumorspheres treated with VC for 48 h. Samples derived from the same experiment and gels/blots were processed in parallel. Data are representative of at least three independent experiments and shown as mean ± s.d. (*p < 0.05; **p < 0.01; ***p < 0.001)
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It is well established that excessive oxidative stress causes depletion of cellular adenosine triphosphate (ATP).31 ATP decreases dependent on time were observed in VC-treated HCC cells and reduction in ATP levels was greater in Huh7 cells expressing higher SVCT-2 than HCC-LM3 cells (Fig. 5e). NAC dramatically reversed VC-induced ATP depletion in HCC cells, suggesting the necessity of ROS in reducing ATP levels (Fig. 5e). Similarly, SVCT-2 silencing also suppressed the depletion of ATP in Huh7 cells following VC treatment in different time points (Fig. 5f). Furthermore, VC induced G2/M phase cell cycle arrest, accompanied by significant reduce in G0/G1 phases and enhanced expression of cyclin-dependent kinase inhibitor p21 in a concentration-dependent manner, consistent with findings with H2O2 and VC-triggered cell cycle arrest was inhibited in the presence of NAC (Fig. 5g and Supplementary Fig. 2f). Knockdown of SVCT-2 remarkably repressed the activation of p21 induced by VC (Supplementary Fig. 2g). Additionally, a characteristic hypodiploid DNA content peak (sub-G1) representing apoptotic cells was detected, indicating VC-induced apoptosis after G2/M arrest (Fig. 5g). Indeed, the proportions of early and late apoptotic cells were significantly increased in a VC concentration-dependent manner (Fig. 5h). Caspase 3 and PARP were cleaved in VC-treated cells (Supplementary Fig. 2f) and the VC-induced decrease in cell viability was partially recovered after pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, implying that VC triggers caspase-dependent death in HCC cells (Fig. 5i and Supplementary Fig. 2e). Cleaved caspase 3 and PARP induced by VC were dramatically reduced in shSVCT-2 cells compared to shCtrl cells, suggesting that VC partially induces caspase-dependent apoptosis in SVCT-2-dependent manner (Supplementary Fig. 2g). Similarly, knocking down SVCT-2 markedly reversed the enhanced expressions of p-H2AX, p21, and cleaved-PARP induced by VC in tumorspheres (Fig. 5j).
We also tested whether VC-induced HCC cell death was dependent on autophagy.32,33,34 The cellular autophagy markers Beclin-1 and LC3B-II proteins were upregulated in VC-treated cells and addition of NAC suppressed expressions of these proteins (Supplementary Fig. 3a), implying that VC is involved in autophagy induction. However, inhibition of autography via an autophagy inhibitor (3-MA) (Supplementary Fig. 3b) or Beclin-1 knockdown had no effect on VC-induced cell death (Supplementary Fig. 3c, d). Therefore, VC triggers autophagy-independent death in human HCC cells. In addition to autophagy and apoptosis, necrosis is another major type of cell death and also functions as an alternative mode of programmed cell death.35,36 To test whether VC induces programmed necrosis or necroptosis, two small compound inhibitors necrostatin-1 (Nec-1) and necrosulfonamide (NSA) were employed to block the activity of central regulators in the programmed necrosis or necroptosis. As a result, neither of the inhibitors alleviated VC-induced cytotoxicity (Supplementary Fig. 3e, f). These results indicate that necroptosis may not be one of the cell death mechanisms triggered by VC. Altogether, these data indicate that VC influx into cells via SVCT-2 and increases intracellular ROS levels, which subsequently induces DNA damage and ATP depletion, leading to cell death partially via cell cycle arrest and caspase-dependent apoptosis, but not autophagy or necroptosis.
Pharmacological VC impairs tumor growth and eradicates liver CSCs in vivo
To further confirm above findings in vivo, we established both HCC cell xenografts and HCC patient-derived xenografts (PDXs) models. Consistent with the in vitro results, stemness-related genes expressions in tumor xenograft were remarkably reduced after VC or VC+cisplatin treatment, whereas conventional cisplatin therapy alone led to the increase of CSCs (Fig. 6b, c). Interestingly, the combination of VC and cisplatin was even more effective in reducing tumor growth and weight (Fig. 6a). Furthermore, either VC or cisplatin alone resulted in increased apoptotic markers expressions, whereas VC and cisplatin combination further caused cell apoptosis in tumor xenograft (Fig. 6b, c). In HCC PDXs models with relative low and high SVCT-2 expression, VC treatment significantly delayed tumor growth (Fig. 6d, e). Intriguingly, PDX#2 and PDX#3, which had relative higher SVCT-2 expression, exhibited lower relative tumor growth and mass compared with PDX#1, suggesting hyper-sensitivity toward VC treatment (Fig. 6d, e). These results verify that VC inhibits tumor growth in HCC PDX models and SVCT-2 expression level is associated with VC response. Furthermore, qPCR and IHC analysis demonstrated that expression levels of CSC-associated genes and percentages of CSCs in PDXs dramatically declined after VC treatment, confirming the inhibitory role of VC in liver CSCs (Fig. 6f, g).
Fig. 6
Pharmacological VC impairs tumor growth and preferentially kills liver CSCs in vivo, and intravenous VC reduces the risk of post-surgical HCC progression. a Huh7 cells were subcutaneously inoculated into nude mice. When tumors grew to ~50 mm3, treatment commenced with intraperitoneal injection of VC (4 g/kg, twice every day) and cisplatin (Cp; 3 mg/kg, twice per week) either alone or in combination. Tumor sizes were measured twice per week. After ~21 days of treatment, mice were euthanized and total tumor weights were measured. b Western blot analysis showing stemness and apoptotic markers expressions in tumor xenograft after treatment of VC and cisplatin either alone or in combination. Samples derived from the same experiment and gels/blots were processed in parallel. c IHC analysis showing stemness and apoptotic markers expressions in tumor xenograft after treatment of VC and cisplatin either alone or in combination. Scale bars = 100 μm. d IHC analysis showing SVCT-2 expression between PDXs from patient #1, #2, and #3. Scale bars = 100 μm. e Relative weights of PDXs from patient #1, #2, and #3 after ~21 days of VC treatment. PDXs were treated intraperitoneally twice daily with either VC (4.0 g/kg) or vehicle (PBS). f qRT-PCR analysis for stemness markers in PDXs from patient #1 and #3 after treatment of either VC or vehicle (PBS). g IHC analysis showing Oct-4, CD133, and Lin28 expressions in PDXs from patient #1 and #3 after treatment of either VC or vehicle (PBS). Scale bars = 100 μm. h DFS of 613 patients with primary HCC after initial hepatectomy receiving 2 g intravenous VC, or not. i Schematic showing how VC kills cancer cells and preferentially kills CSCs via SVCT-2. Data are representative of at least three independent experiments and shown as mean ± s.d. (*p < 0.05; **p < 0.01; ***p < 0.001)
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Intravenous VC reduces the risk of post-surgical HCC progression
Liver protection treatment is regularly given to HCC patients after hepatectomy. VC is one of the numerous common hepatoprotectants.37 In our Eastern Hepatobiliary Surgery Hospital, Shanghai, China, some HCC patients received intravenous VC after hepatectomy. Pharmacokinetics studies in human show that 2 g of intravenous VC achieves a plasma concentration of nearly 1.5 mM.10 Interestingly, at extracellular concentrations greater than 1 mM, VC induces strong cytotoxicity to cancer cells including liver cancer cells, as demonstrated in the above studies.38 Therefore, we hypothesized that intravenous VC might reduce the risk of recurrence in HCC patients after curative liver resection.
Six hundred thirteen HCC patients who received curative liver resection as their initial treatment between 2008 and 2009 and met the inclusion criteria were enrolled in the analyses. HCC patients were divided into two groups: VC users and non-VC users. Three hundred thirty-nine participants (55.3%) received 2 g intravenous VC for 4 or more days after initial hepatectomy. As shown in Supplementary Table 2, the distribution of clinicopathologic factors between VC users and non-users was no significant difference. Intriguingly, the 5-year disease-free survival (DFS) for patients who received intravenous VC was 24%, as opposed to 15% for no intravenous VC-treated patients (p < 0.001) (Fig. 6h). Median DFS time for VC users was 25.2 vs. 18 months for VC non-users (p < 0.001). Univariate analysis revealed that tumor size ≥5 cm, multiple tumor numbers, AFP ≥ 20 μg/L, AFP ≥ 400 μg/L, tumor thrombus, and no post-surgical intravenous VC administration were significantly associated with shorter DFS (Table 1). Multivariate analysis further demonstrated that intravenous VC administration was an independent factor for improved DFS (adjusted HR = 0.622, 95% CI 0.487 to 0.795, p < 0.001) (Table 1). These results suggest that intravenous VC use is linked to improved DFS in HCC patients.
Table 1 Univariate/multivariate analysis of prognostic factors associated with the DFS of 613 HCC patients
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Discussion
Despite the recent advances in liver cancer therapy, it remains one of the most lethal malignancies. VC has a controversial history in cancer treatment. In the 1970s, Pauling and Cameron reported that intravenous VC (10 g/day) was effective in prolonging the survival of cancer patients.5,6,7 However, clinical trials performed by Mayo Clinic found the same dose of VC ineffective in treating cancer by using it orally.8,9 It was recognized later that the route of VC administration was the main reason for the discrepancy. Pharmacologic concentrations of plasma VC, which are achievable only from i.v. administration other than oral VC, can kill cancer cells.10 Currently, pharmacologic VC has garnered increased interest in the field of cancer therapy. However, few studies have investigated the effect of VC on CSCs, the subpopulation responsible for tumor initiation, metastasis, recurrence, and resistance to chemotherapy.3,4 In this study, based on the elevated expression of SVCT-2, which is responsible for VC uptake, in liver CSCs, we revealed that clinically achievable concentrations of VC preferentially eradicated liver CSCs in vitro and in vivo. Additionally, we found that intravenous VC reduced the risk of post-surgical HCC progression in a retrospective cohort study.
As the key protein responsible for VC uptake in the liver, SVCT-2 played crucial roles in regulating the sensitivity to ascorbate-induced cytotoxicity.34 In this study, we also revealed that SVCT-2 expressions were inversely associated with IC50 values of VC and positively correlated with intracellular VC concentrations in HCC cells after VC treatment. Conversely, SVCT-2 silencing in Huh7 cells dramatically decreased the sensitivity to VC. Strikingly, we also observed that SVCT-2 was highly expressed in human HCC samples and preferentially elevated in liver CSCs. Knocking down SVCT-2 expression significantly affected self-renewal, chemoresistance, and tumorigenicity of liver CSCs. In this regard, SVCT-2 might serve as a potential CSC marker and therapeutic target in HCC. Unexpectedly, physiological concentration of VC does not markedly promote HCC in vitro. We found that low dose (0.1 mM) of VC had no significant influence on HCC cells growth and the stemness-related genes expressions (Supplementary Fig. 4a, b). Nevertheless, our in vitro conditions are unable to sufficiently mimic the in vivo environment with hypoxia, hypoglycemia, and other metabolic changes. Therefore, further studies are needed to evaluate the effect of physiological VC on HCC in vitro and in vivo.
CSCs play critical roles in regulating tumor initiation, relapse, and chemoresistance.3,4 In HCC, we have previously demonstrated that OV6+ liver CSCs exhibit resistance to chemotherapy and contribute to HCC progression and invasion.26,27 Contrary to expectations, VC is distinguished from other well-defined chemotherapeutic drug (e.g., cisplatin, doxorubicin) and VC treatment does not lead to the enrichment of CSCs. Instead, by detecting key features of CSCs in vitro and in vivo, we revealed that VC treatment dramatically reduced the self-renewal ability, expression levels of CSC-associated genes, and percentages of CSCs in HCC, indicating that CSCs were more susceptible to VC-induced cell death. Thus, as a drug for eradicating CSCs, VC may represent a promising strategy for treatment of HCC, alone or particularly in combination with chemotherapeutic drugs.
It is accepted that the cytotoxicity of pharmacologic VC is mediated by generation of sustainable ascorbate radical and H2O2.11 However, there is no general molecular mechanism suitable for heterogeneous cancer cells because H2O2 could produce downstream ROS and influence various cellular and molecular targets. Previous studies have reported multiple mechanisms in different cancers, including caspase-dependent and caspase-independent apoptosis,39 nonapoptotic cell death,11,40 autophagy, 16 autoschizis,41 ATP depletion,25 DNA damage,25,42 and cell cycle arrest.42 In HCC, we found that VC-generated ROS caused genotoxic stress (DNA damage) and metabolic stress (ATP depletion), which further activated the cyclin-dependent kinase inhibitor p21, leading to G2/M phase cell cycle arrest and caspase-dependent apoptosis in HCC cells (Fig. 6i). Furthermore, we demonstrated a synergistic effect of VC and chemotherapeutic drug cisplatin on killing HCC both in vitro and in vivo. It is known that cisplatin treatment also results in DNA damage despite through a distinct mechanism from that in VC.43 Cisplatin induces DNA damage via reaction of the platinum molecule with nucleophilic sites rather than ROS.43 As a result, VC and cisplatin combination led to larger extent of DNA damage in HCC cells than either use alone. Intravenous VC has also been reported to reduce chemotherapy-associated toxicity of carboplatin and paclitaxel in patients,38 but the specific mechanism needs further investigation.
Pharmacokinetics studies show that 2 g of intravenous VC achieves a plasma concentration of nearly 1.5 mM,10 a concentration sufficient to induce death in HCC cells, as evidenced by our in vitro studies. Our retrospective cohort study also showed that intravenous VC use (2 g) was related to the improved DFS in HCC patients after initial hepatectomy. In fact, several clinical trials of high-dose intravenous VC have been conducted in patients with advanced cancer and have revealed improved quality of life and prolonged OS.44 Considering the much higher dose (≥50 g) employed in these clinical trials, additional clinical trials will be needed to prove the safety, efficacy, and doses of VC in HCC treatment. All xenografts were performed in nude mice with compromised immune system to test the anti-tumor effect of VC in the above studies. Since VC may help boost body immune system to fight against cancer, we further examined the effect of high-dose VC on HCC progression and immune cells using normal mice. Similarly, VC treatment significantly inhibited growths of tumors derived from mouse liver cancer cells (Hepa1-6) in C57BL/6 mouse (Supplementary Fig. 4c, d). Furthermore, high-dose VC was not toxic to immune cells and major immune cell subpopulations in vivo (Supplementary Fig. 4e, f). Thus, the inhibitory effect of pharmacologic VC on liver cancer may be not mainly through the promotion of immune system. Taken together, our findings unravel the potential application of VC for HCC therapy. The mechanisms about how pharmacologic VC kills cancer cells and preferentially kills CSCs via SVCT-2 are summarized in Fig. 6i. Notably, we also propose that SVCT-2 is a new CSC marker and therapeutic target in HCC and its expression level may serve as a biomarker for VC response.
Methods
Patients
In the retrospective study, a total of 669 patients with primary HCC who underwent initial curative liver resection in the Eastern Hepatobiliary Surgery Hospital, Shanghai, China, from 2008 to 2009 were collected. Of these, 613 patients who met the inclusion criteria were finally enrolled. The inclusion criteria included: (1) the diagnosis of HCC was based on World Health Organization criteria; (2) none of the patients received chemotherapy or radiotherapy before the surgery. HCC patients were divided into two groups: VC users (n = 339) and VC non-users (n = 274). Patients who received 2 g intravenous VC for 4 or more days after initial hepatectomy were defined as VC users. The clinicopathological features of 613 patients were summarized in Supplementary Table 2. Additionally, a tissue microarray composed of HCC samples from 104 patients used to examine the prognostic significance of SVCT-2 expression (Fig. 1b, c) was obtained from the Eastern Hepatobiliary Surgery Hospital. The clinicopathological features of 104 patients were summarized in Supplementary Table 1. Another 19 fresh HCC tissues were also obtained from the Eastern Hepatobiliary Surgery Hospital to evaluate the correlation between SVCT-2 and stemness-related genes by qRT-PCR analysis (Fig. 1d). Patient consent was obtained prior to the start of the study. All studies were approved by the Ethical Committee of the Second Military Medical University (SMMU) and performed in accordance with relevant regulations and guidelines.
In vivo xenograft assay
1 × 106 shCtrl, shSVCT-2-1, and shSVCT-2-2 Huh7 cells were injected subcutaneously into the right flank of each male nude mouse (Chinese Science Academy, Shanghai, China). To investigate the role of VC in cancer treatment in vivo, 1 × 106 human HCC cell lines (HCC-LM3 and Huh7) were injected subcutaneously into the right flank of each nude mouse and 1 × 106 mouse liver cancer cell line (Hepa1-6) was injected subcutaneously into the right flank of each male C57BL/6 mouse (Chinese Science Academy, Shanghai, China). When tumors grew to ~50 mm3, mice were randomized into two groups (n = 6) and treatment commenced with intraperitoneal injection of 4 g/kg VC (equivalent to ~1.3 g/kg i.v.),12 a dose widely used in numerous studies to test the effect pharmacological VC on various cancer treatment in mouse model,13,16,38 or vehicle (PBS) twice every day for ~21 days. In another study, 1 × 106 Huh7 cells were injected subcutaneously into the right flank of each nude mouse. When tumor volume had reached ~50 mm3, mice were randomized into four groups (n = 6) and treatment commenced with intraperitoneal injection as follows: (i) Ctrl, PBS twice daily; (ii) VC, vitamin C at 4 g/kg twice daily; (iii) Cp, cisplatin at 3 mg/kg twice per week; (iv) VC+Cp.
For HCC PDX model, fresh tumor specimens were procured from previously established PDX models (passage 2–3) and cut into small tissue blocks (~50 mm3) before engrafted subcutaneously into male nude mice (Chinese Science Academy, Shanghai, China). After 2–3 weeks, PDXs from patient #1 (n = 6) and patient #2 (n = 6) were intraperitoneally treated with either VC (4 g/kg) or vehicle (PBS) twice daily. Tumor size (length × width2 × 0.5) was measured twice per week after treatment. At ~25 days, all mice were euthanized and tumors were excised and weighed. Mice were employed between 4 and 6 weeks of age and the number of mice per group was selected to provide sufficient statistical power to the experiment based on the expected biological variation. Investigators were not blinded as to group allocation. All animal experiments were approved by the Ethical Committee of the SMMU and performed in accordance with relevant regulations and guidelines.
Statistics
Statistical analysis was carried out using SPSS 22.0 software (SPSS Inc., USA). The data are presented as the mean ± s.d. Two-tailed Student's t-test was used to determine the significance of differences between groups. Pearson's correlation analysis was applied to determine the correlation between two variables. The survival rate was calculated using the Kaplan–Meier method and univariate survival analysis was done by the log-rank test. Multivariate analysis was performed using the Cox proportional hazards model. p-value < 0.05 was considered as significant.
Additional methods are described in Supplementary Information.
Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information files.
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Acknowledgements
The authors are grateful to Liang Tang, Linna Guo, Dan Cao, Shanna Huang, Dongping Hu, Shanhua Tang, and Dandan Huang for their technical assistances. This work was supported by National Natural Science Foundation of China (81622039, 81221061, 81572895, 81372356).
Author information
Author notes
Hongwei Lv, Changzheng Wang, and Tian Fang contributed equally to this work.
Affiliations
International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, 200438, Shanghai, China
Hongwei Lv, Changzheng Wang, Tian Fang, Ting Li, Guishuai Lv, Qin Han, Wen Yang & Hongyang Wang
The Fifth Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 200438, Shanghai, China
Changzheng Wang
National Center for Liver Cancer, 201805, Shanghai, China
Guishuai Lv, Wen Yang & Hongyang Wang
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University, 200032, Shanghai, China
Hongyang Wang
Contributions
H.L., C.W., T.F., T.L., G.L., and Q.H. performed the experiments and H.L., C.W., T.F., W.Y., and H.W. analyzed the data. W.Y. and H.W. designed the project. H.L., W.Y., and H.W. analyzed the data and wrote the manuscript. H.L., W.Y., and H.W. revised the manuscript.
Corresponding authors
Correspondence to Wen Yang or Hongyang Wang.
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Lv, H., Wang, C., Fang, T. et al. Vitamin C preferentially kills cancer stem cells in hepatocellular carcinoma via SVCT-2. npj Precision Onc2, 1 (2018). https://doi.org/10.1038/s41698-017-0044-8
