Ovarian cancer is a common and deadly type of cancer which often develops resistance to drugs such as cisplatin, a barrier to its treatment. The cisplatin resistance arises from undesirable binding of the drug to glutathione and metallothionein, thus inactivating it. Pradip Mascharak, Distinguished Professor of Chemistry and Biochemistry at the University of California, Santa Cruz, has shown that in ovarian cancer cells, cisplatin resistance can be reduced with targeted delivery of carbon monoxide (CO) via photoCORMs as CO donors. Carbon monoxide produced this effect by inhibiting cystathionine β-synthase, an enzyme involved in the synthesis of glutathione and metallothionein.
According to the Centers for Disease Control and Prevention (CDC), ovarian cancer is the United States’ second most common gynaecologic cancer, causing more deaths than any other cancer of this bodily region. In 2020, it was estimated that there were 21,750 new cases of ovarian cancer in the U.S., resulting in 13,940 deaths. This type of cancer has a 5-year relative survival rate of 48.6%.
One major factor limiting treatment of ovarian cancer is the cancer cells becoming resistant to common drugs, such as cisplatin. There is clearly an unmet need for methods to help overcome this barrier, making previously resistant cancers treatable with chemotherapy. Luckily, there may be an unlikely hero in this scenario: carbon monoxide. A team at University of California, Santa Cruz led by Prof Pradip Mascharak have published a study explaining how a method of targeting carbon monoxide to drug-resistant ovarian cancer cells could lower their drug resistance. This could lead to better, more effective treatment for this common and deadly cancer.
How does carbon monoxide function in the body?
Carbon monoxide (CO) is a gas that has no smell, colour, or taste. It is usually formed by the incomplete combustion of compounds containing carbon, commonly in fires, faulty furnaces, and engine exhausts. There is no known antidote to CO poisoning. Haemoglobin carries oxygen around the body and has a much higher binding affinity to CO than oxygen – 250 times higher, in fact. This competition for binding reduces the amount of oxygen bound to haemoglobin. In addition to reducing haemoglobin-oxygen binding, CO also inhibits respiration in the mitochondria. It does this by binding to cytochrome c oxygenase, which is vital for oxidative phosphorylation, a key process for the generation of energy in cells. This, along with other processes, leads to CO causing the formation of reactive oxygen species that can damage cells.
How carbon monoxide can be used in cancer treatment
Despite the potentially fatal effects of CO, it can have some positive effects in the right circumstances. Low concentrations of CO have been shown to improve organ and graft survival rates in animals, as well as protect patients against ischemic reperfusion injury after cardiopulmonary bypass surgery. Low doses of CO also promote programmed cell death in hyperproliferating cancer cells of various cancer types. It can also make resistant cancer cells more sensitive to chemotherapy and help destroy cancer cells that are malignant.
The toxicity of CO gas, however, has led to difficulty handling it, thus limiting its medical use. This means that there is a demand for molecules that release CO in a controlled manner to a target. This has resulted in the development of photoactive CO-releasing molecules, or photoCORMS. These are metal carbonyl complexes that only release CO when triggered by specific wavelengths of light. In a paper recently published by Prof Mascharak, the research team has investigated the use of photoCORM treatment on ovarian cancer cell lines resistant to the cancer drug cisplatin.
What did the study find?
The researchers started out by investigating the effect of CO delivery via photoCORM to ovarian cancer cell lines modified to be cisplatin resistant. The cell lines were called OVcisR and SKVcisR. They were administered cisplatin, either alone or alongside photoCORM. With cisplatin alone, viability of OVcisR and SKVcisR decreased by around 40% and 29%. When accompanied by photoCORM, however, this decrease in viability was doubled. This effect was probably down to the CO rather than the photoCORM scaffold, as no effect was observed when there was no light to activate CO release.
With the addition of N-acetylcysteine (NAC), an antioxidant and cysteine donor, this sensitising effect of CO was reversed. The addition corresponded with increased levels of cysteine within the cells, suggesting that cysteine levels may have a role in resistance to cisplatin.
One major source of cysteine for cells is the transsulfuration pathway, which involves the action of two enzymes, namely cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL). The first step is catalysed by CBS, breaking down homocysteine to cystathionine (CTH). CTH is then broken down by CGL into cysteine and other compounds. In this study, it was observed that the cisplatin-resistant cell lines had dramatically more CBS, CGL, and CTH than non-resistant lines.
When the expression of CBS was silenced, thus reducing its production, previously resistant cell lines became more sensitive to cisplatin treatment. A drug’s ED50 is the amount needed to produce the desired effect in half of the cells. Resistant OVcisR and SKVcisR had ED50s around 11 μM and 30 μM, whereas CBS-silenced OVcisR and SKVcisR had lower ED50s, around 2.5μM and 3.6μM. In CBS-silenced cells, cysteine levels were also lower. CBS-silenced OVcisR and SKVcisR had around 60% and 70% lower levels of cysteine than their non-silenced counterparts.
Another significant source of cysteine for cancer cells is the xCT transporter. This transporter brings cystine into the cell, which is two cysteines linked together. Cystine is quickly converted to cysteine within the cell. The researchers labelled cystine with deuterium, an isotope of hydrogen, to measure its uptake in cisplatin-resistant and non-resistant cells. The expression of xCT was not much different between the two types. However, levels of cysteine containing deuterium were around 2.1 times higher in OVcisR and 3.4 times higher in SKVcisR compared to their non-resistant counterparts, indicating a greater cystine uptake capacity. Uptake of cystine halved when CBS was silenced. This could be explained by H2S, a possible product of CBS that increases xCT activity.
Cisplatin being bound and inactivated by glutathione (GSH) and metallothionein (MT) is a cause of cisplatin resistance. GSH and MT both contain cysteine. Levels of GSH and MT were examined and were found to be higher in the resistant cell lines compared to non-resistant. The levels of GSH and MT increased even further with NAC treatment. CBS-silencing reduced GSH levels by over 50%, and also reduced the amount of MT in the nucleus.
The researchers finished by investigating whether CO increasing sensitivity to cisplatin in previously resistant cells could be down to inhibition of CBS, and therefore suppression of MT and GSH. The cisplatin-resistant cell lines were treated again with photoCORM. They found that treatment with photoCORM significantly lowered the activity of CBS, significantly reduced cystine uptake, and reduced levels of cysteine by over 55%. It was also shown that cells treated with photoCORM had lower levels of GSH, and lower levels of MT in the nucleus.
What does this mean?
The data from this study are supported by the results of previous studies led by Prof Mascharak on breast cancer cells. The authors mention that the results “require critical evaluation”; however, they have big implications. Inhibition of CBS via pharmacological agents has not yet been achieved. Even if it had been, CBS antioxidant activity in the liver is important, so drugs that affect the whole body rather than just the cancer could have bad side effects. This study highlights photoCORMs as CBS inhibitors suitable for clinical use, as they are effective and only active at locations where they are activated by light. With further study, photoCORM use could be a breakthrough in tackling cisplatin-resistant cancers. This could lead to carbon monoxide, previously known as a silent killer, helping to save lives.
- Kawahara, B., Ramadoss, S., Chaudhuri, G., Janzen, C., Sen, S., Mascharak, P. K. (2019). Carbon monoxide sensitizes cisplatin-resistant ovarian cancer cell lines toward cisplatin via attenuation of levels of glutathione and nuclear metallothionein. Journal of Inorganic Biochemistry, 191, 29–39. Available at: https://www.sciencedirect.com/science/article/pii/S0162013418303362?via=ihub
- Rose, J., Wang, L., Xu, Q., McTiernan, C., Shiva, S., Tejero, J. and Gladwin, M. (2017). Carbon Monoxide Poisoning: Pathogenesis, Management, and Future Directions of Therapy. American Journal of Respiratory and Critical Care Medicine, 195(5), 596–606. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363978/
- Carrington, S. J., Chakraborty, I., Bernard, J. M., & Mascharak, P. K. (2014). Synthesis and Characterization of a “Turn-On” photoCORM for Trackable CO Delivery to Biological Targets. ACS medicinal chemistry letters, 5(12), 1324–1328. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265820/
- CDC (2020). Ovarian Cancer Statistics. [online] cdc.gov. Available at: https://www.cdc.gov/cancer/ovarian/statistics/index.htm [Accessed 03 01 2021]
- National Cancer Institute. Cancer Stat Facts: Ovarian Cancer. [online] National Cancer Institute Surveillance, Epidemiology, and End Results Program. Available at: https://seer.cancer.gov/statfacts/html/ovary.html [Accessed 03 01 2021]
Prof Pradip Mascharak investigates the effects of carbon monoxide in circumventing chemotherapeutic drug resistance.
Pradip Mascharak, Distinguished Professor of Chemistry and Biochemistry at the University of California, Santa Cruz, is a bioinorganic chemist. He received his PhD from the Indian Institute of Technology at Kanpur, India, in 1978 and did his post-doctoral work first at Stanford University and then at Harvard University. He also worked as a research associate at Massachusetts Institute of Technology for two years before he joined the Department of Chemistry and Biochemistry in 1984.
Department of Chemistry and Biochemistry
University of California
Santa Cruz, CA 95060
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