Cancer addiction to Cyst(e)ine & Lowering Glutathione inside cancer cells

Recently, I came across this nice article (Ref) which is in turn based on a recent publication (Ref.) arguing that in some types of renal cancer cells, cystine deprivation “presents an attractive therapeutic opportunity that may bypass the apoptosis-evading mechanisms characteristic of drug-resistant tumor cells.” However, as we will see Cysteine is relevant not only in renal cancer but in most of the other cancers, and Cysteine deprivation strategy may work well with many treatments including chemo.

The final and main goal of Cysteine deprivation is to reduce the level of anti oxidants in the tumors which are usually high and are used by the tumors as a drug resistance mechanism to fight anti-cancer treatments.

What is the role of Cystine/Cysteine

Cystine is converted to Cysteine, and Cysteine is the fuel for Glutathione  production: Cysteine is (a sulfur-containing amino acid and) an important structural and functional component of proteins and enzymes. Glutathione is probably the most important anti oxidant in the body, and Cysteine is one of its building blocks. Specifically, the body requires three amino acids – glutamate, glycine and cysteine – in order to manufacture glutathione on its own inside the cell. This means that the level of cysteine in your system is the limiting factor in how fast you can produce glutathione and how much of it you can make. Our body produces some cysteine on its own from another amino acid – methionine. Methionine is an essential amino acid, meaning it is not produced by the body but comes from diet only (from high-protein foods: all meats and poultry, dairy and eggs, quinoa, buckwheat). (Ref.)

Glutathione helps cancer cells to survive pro oxidant treatments like chemo: Glutathione is a major antioxidant considered essential for protection of cells from oxidative stress. It plays an important role as a detoxifier and is believed to be on of the main elements responsible for drug resistance of cancer cells to therapies. Oxidative stress is particularly generated in cancer cells because of their relatively high metabolism, and glutathione is responsible for helping cancer cells to survive that oxidative stress. On this line, glutathione  depletion has been suggested as a therapeutic approach for a variety of cancers, in particular to reduce resistance to conventional anticancer agents (Ref). Indeed, in general chemo therapies contribute to this oxidative stress in cancer cells that is managed by glutathione  and this is also why, the conventional medicine suggests to the patients not to use strong anti oxidants while being treated with chemo substances.

In conclusion, Cysteine is a rate-limiting precursor of Glutathione, and sustenance of adequate intracellular cysteine levels is critical for maintaining adequate Glutathione levels, cell growth, and viability. For some cancers, this sustenance depends on uptake of cysteine or cystine (the oxidized form of the amino acid) from the microenvironment.

Cystine/Cysteine transporters are over-expressed in cancer:  Indeed, it has been indicated that stromal cells (for example, fibroblasts) which are the neighbors and helpers of cancer cells are producing Cysteine that is being taken up by cancer cells (using the ubiquitous ASC transport system). In order to produce Cysteine, the stromal cells are taking up Cystine via plasma membrane cystine/glutamate antiporter (Ref). Therefore, acting as feeder layers, the fibroblasts convert Cystine to Cysteine and supply cysteine (essential for growth and resistance to treatments) to cancer cells.

Besides fibroblasts cancer cells seem to also have the cystine/glutamate transporter overexpressed relative to normal. (Ref1, Ref2)

Interestingly, the absorption of cystein and the export of glutamate not only helps cancer cells but also leads to pain which means that inhibiting this process with a drug such as the one discussed below, will lead to pain reduction in cancer patients (Ref.).

How to use this knowledge in cancer treatment:
Cysteine starvation by inhibiting relevant transporters

On this line, inhibition of the cystine/glutamate transporter, leading to cystine/cysteine starvation and subsequent glutathione depletion, has been proposed as a potential therapeutic approach in a variety of cancers (Ref).

Indeed, Sulfasalazine, a cystine/glutamate transporter inhibitor has been indicated  to be relevant for targeted therapy in various cancers such as

• pancreatic cancer http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2880911/
• lymphoma http://www.nature.com/leu/journal/v15/n10/full/2402238a.html
• gastric cancer http://meetinglibrary.asco.org/content/134696-144
• triple negative breast cancer https://www.ucsf.edu/news/2013/10/109376/scientists-identify-triple-negative-breast-cancer-target-drug-development
• brain cancer http://www.tandfonline.com/doi/abs/10.1517/13543784.2012.670634 (However, note that here is a clinical trial in glioma showing no response to Sulfasalazine, yet no cotreatment has been used http://bmccancer.biomedcentral.com/articles/10.1186/1471-2407-9-372 and than here is a recent study showing that when used together with difluoromethylornithine may be very effective against neuroblastoma http://msutoday.msu.edu/news/2015/two-common-medicines-found-to-fight-deadly-childhood-cancer/)
• bone cancer and mets http://www.sciencedirect.com/science/article/pii/S0304395913004934
• colorectal cancer http://www.sciencedirect.com/science/article/pii/S0304383515004863
• prostate cancer https://www.researchgate.net/publication/6720301_Sulfasalazine-induced_cystine_starvation_Potential_use_for_prostate_cancer_therapy
• lung cancer http://cancerres.aacrjournals.org/content/64/7_Supplement/887.2.short http://www.ncbi.nlm.nih.gov/pubmed/17440102

Sulfasalazine is a FDA approved drug used in the treatment of inflammatory bowel disease, including ulcerative colitis and Crohn’s disease. It is also indicated for use in rheumatoid arthritis and used in other types of inflammatory arthritis (e.g. psoriatic arthritis) where it has a beneficial effect. (Ref)

Overall, the response to Sulfasalazine does not appear to be tissue or organ specific, suggesting this is a common cellular mechanisms.

Administration:

Sulfasalazine:

• 8g/day in a clinical trial http://meetinglibrary.asco.org/content/134696-144
• other studies indicated 2-6g/day
• some patients on Cancer Compass using it in combination with other theraphies http://www.cancercompass.com/message-board/message/all,3125,1.htm
• usually taken four times/day

This seems to be a good approach when combining with other (pro oxidant) therapies such as chemo, DCA, 3BP and others. I would probably us it during Chemo or other pro oxidant therapists and would stop it from time to time so that the non cancerous part of the body will receive its Glutathione and detoxify. I would also probably not take more than 2.5g/day just to be on the safe side.

Synergy and Antagonists

Most of the chemotheraphies and radiotheraphy act via pro oxidant mechanisms so Sulfasalazine should help them. It should also work well with the 3BP treatment and DCA. Both of these elements are known to work well when the intracellular Glutathione level is reduced (Ref.)

Avoid NAC supplements that will antagonize Sulfasalazine.

Source:

http://www.buy-pharma.co/Generic-Azulfidine-Sulfasalazine-Tablet-p-154.html

References:

Increased Oxidative Stress as a Selective Anticancer Therapy http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529973/

Reactive oxygen species (ROS) are closely related to tumorgenesis. Under hypoxic environment, increased levels of ROS induce the expression of hypoxia inducible factors (HIFs) in cancer stem cells (CSCs), resulting in the promotion of the upregulation of CSC markers, and the reduction of intracellular ROS level, thus facilitating CSCs survival and proliferation. Although the ROS level is regulated by powerful antioxidant defense mechanisms in cancer cells, it is observed to remain higher than that in normal cells. Cancer cells may be more sensitive than normal cells to the accumulation of ROS; consequently, it is supposed that increased oxidative stress by exogenous ROS generation therapy has an effect on selectively killing cancer cells without affecting normal cells. This paper reviews the mechanisms of redox regulation in CSCs and the pivotal role of ROS in anticancer treatment.

Sulfasalazine: Potential use of an old drug for treatment of pancreatic cancer http://cancerres.aacrjournals.org/content/66/8_Supplement/1105.4

Pancreatic cancer is one of the most aggressive and therapy-resistant cancers known. Although chemotherapy using gemcitabine has yielded increases in patient survival, there is no effective therapy for this disease. We have previously found that sulfasalazine (SASP), an established anti-inflammatory drug, can arrest proliferation of lymphoma and prostate cancer cells both in vitro at patient-tolerated levels (∼0.2 mM) and in experimental animals. The in vitro action of SASP is largely based on inhibition of cystine uptake via the x_c^– cystine/glutamate antiporter. This amino acid, essential for cell growth and viability, cannot be synthesized by certain cancers, rendering them dependent on its uptake from their environment. In the present study we investigated the potential usefulness of SASP for therapy of pancreatic cancer. Human pancreatic cancer cell lines, Mia PaCa-2, Panc-1 and Bxpc-3, were incubated with SASP at a range of concentrations for various periods and cell numbers determined via the WST-1 cell proliferation assay. RT-PCR was used to determine mRNA expression of the xCT subunit of the x_c^– cystine transporter. In vivoactivity of SASP was evaluated using Rag-2M mice carrying subrenal capsule xenografts of the cancer cells (about 80 mm³). I.p. injections of saline or SASP (250 mg/kg body weight) were administered every 12 h for 7 days; the effect of gemcitabine (120 mg/kg body weight; i.p.), twice a week for 7 days, was also assessed. Animals were then sacrificed and tumors harvested for volume measurement and histological analysis. In vitro, Mia PaCa-2 and Panc-1 cells were particularly sensitive to growth inhibition and lytic action by SASP (IC₅₀ = 0.05 mM and 0.1 mM, respectively); the inhibition could be prevented by 60 μM 2-mercaptoethanol allowing cystine uptake via the leucine transporter. The Bxpc-3 cells were less sensitive to SASP (IC₅₀ = 0.35 mM), showing increased expression of x_c^–, as indicated by elevated xCT-mRNA levels. In vivo, both SASP and gemcitabine inhibited growth of xenografts of the three cell lines, showing growth arrest of about 40-60% (relative to controls), without major toxicity to the hosts. While the growth-inhibitory effect of SASP on pancreatic cancer cell proliferation in vitro involves cystine starvation, the anticancer activity of SASP in vivo needs further investigation. Nevertheless, the marked anti-tumor activity of SASP without major toxicity to the hosts suggests that this established drug could be useful in combination chemotherapy of pancreatic cancer.

Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the xc- cystine transporter: a new action for an old drug http://www.nature.com/leu/journal/v15/n10/full/2402238a.html

Although cyst(e)ine is nutritionally a non-essential amino acid, lymphoid cells cannot synthesize it, rendering their growth dependent on uptake of cyst(e)ine from their micro-environment. Accordingly, we previously suggested that the x_c^– plasma membrane cystine transporter provided a target for lymphoid cancer therapy. Its inhibition could lead to cyst(e)ine deficiency in lymphoma cells via reduction of both their cystine uptake and cysteine supply by somatic cells. In this study, using rat Nb2 lymphoma cultures, drugs were screened for growth arrest based on x_c^– inhibition. Sulfasalazine was fortuitously found to be a novel, potent inhibitor of the x_c^– transporter. It showed high rat lymphoma growth-inhibitory and lytic activity in vitro (IC₅₀ = 0.16 mM), based specifically on inhibition of x_c^–-mediated cystine uptake, in contrast to its colonic metabolites, sulfapyridine and 5-aminosalicylic acid. Sulfasalazine was even more effective against human non-Hodgkin’s lymphoma (DoHH2) cultures. In rats (n = 13), sulfasalazine (i.p.) markedly inhibited growth of well-developed, rapidly growing rat Nb2 lymphoma transplants without apparent side-effects. Reduced, macrophage-mediated supply of cysteine was probably involved. In five rats, 90–100% tumor growth suppression, relative to controls, was obtained. The x_c^– cystine transporter represents a novel target for sulfasalazine-like drugs with high potential for application in therapy of lymphoblastic and other malignancies dependent on extracellular cyst(e)ine.

Effect of sulfasalazine (SSZ) on cancer stem-like cells (CSCs) via inhibiting xCT signal pathway: Phase 1 study in patients with gastric cancer (EPOC 1205). http://meetinglibrary.asco.org/content/134696-144

Background: CD44 is an adhesion molecule expressed in cancer stem-like cells (CSCs). Our group recently reported that CD44 splice variant (CD44v) is expressed in CSCs and interacts with xCT, a glutamate-cystine transporter, keeping high levels of the intracellular reduced glutathione (GSH). Thus, CSCs with a high expression of CD44v have an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS), resulting in resistance to various therapeutic stresses. Sulfasalazine (SSZ) as an xCT inhibitor suppressed CD44v-dependent tumor growth and increased sensitivity to cytotoxic drugs in vivo study. Methods: A phase 1 dose escalation study in patients with advanced gastric cancer was conducted to determine the optimal dose. SSZ was given fourth-daily oral administration with 2 weeks as one cycle. A 3+3 escalation was used to evaluate a MTD. Tumor tissues were obtained pre- and post SSZ administration to evaluate expression of CD44v and intra-tumor level of GSH by immunohistochemistry and boron doped diamond microelectrode, respectively. Results: Eleven patients were dosed from 8 g to 12 g/day; median age: 71 years (61-78); median number of prior chemotherapies: 3 (1-4). There was two DLT of grade 3 anorexia and nausea among patients who were treated with 12 g/day. One additional patients required frequent dose interruption with grade 2 anorexia and nausea. Therefore 12g/day was judged as MTD. No DLT was observed among patients with 8g/day. Patients with high CD44v expression patientss achieved reduced expression of CD44v after the administration of SSZ for 2 weeks as well as decreased level of GSH. The individual variability of SSZ exposure was explainable in terms of the genotypes of ABCG2 and NAT2 which influence SSZ pharmacokinetics. Conclusions: Optimal dose of SSZ was considered as 8g/day. Down regulation of CD44v expression and decreased level of GSH migh be a pharmacodynamic marker of drug-on-target effect and mode of action of SSZ for CSCs, which warrants further investigation for combination with chemotherapy or other targeting agents. Clinical trial information: UMIN000010254.

Inhibition of breast cancer-cell glutamate release with sulfasalazine limits cancer-induced bone pain http://www.sciencedirect.com/science/article/pii/S0304395913004934

Cancer in bone is frequently a result of metastases from distant sites, particularly from the breast, lung, and prostate. Pain is a common and often severe pathological feature of cancers in bone, and is a significant impediment to the maintenance of quality of life of patients living with bone metastases. Cancer cell lines have been demonstrated to release significant amounts of the neurotransmitter and cell-signalling molecule l-glutamate via the system x_C⁻ cystine/glutamate antiporter. We have developed a novel mouse model of breast cancer bone metastases to investigate the impact of inhibiting cancer cell glutamate transporters on nociceptive behaviour. Immunodeficient mice were inoculated intrafemorally with the human breast adenocarcinoma cell line MDA-MB-231, then treated 14 days later via mini-osmotic pumps inserted intraperitoneally with sulfasalazine, (S)-4-carboxyphenylglycine, or vehicle. Both sulfasalazine and (S)-4-carboxyphenylglycine attenuated in vitro cancer cell glutamate release in a dose-dependent manner via the system x_C⁻ transporter. Animals treated with sulfasalazine displayed reduced nociceptive behaviours and an extended time until the onset of behavioural evidence of pain. Animals treated with a lower dose of (S)-4-carboxyphenylglycine did not display this reduction in nociceptive behaviour. These results suggest that a reduction in glutamate secretion from cancers in bone with the system x_C⁻ inhibitor sulfasalazine may provide some benefit for treating the often severe and intractable pain associated with bone metastases.

Xc− inhibitor sulfasalazine sensitizes colorectal cancer to cisplatin by a GSH-dependent mechanism http://www.sciencedirect.com/science/article/pii/S0304383515004863

Sulfasalazine (SSZ) is an anti-inflammatory drug that has been demonstrated to induce apoptosis and tumor regression through inhibition of plasma membrane cystine transporter xc⁻. Cysteine is a rate-limiting precursor for intracellular glutathione (GSH) synthesis, which is vital for compound detoxification and maintaining redox balance. Platinum-based chemotherapy is an important regimen used in clinics for various cancers including colorectal cancer (CRC). We hypothesized that targeting xc⁻transporter by SSZ may annihilate cellular detoxification through interruption of GSH synthesis and may enhance the anti-cancer activity of cisplatin (CDDP) by increasing drug transport. In the present study, we revealed that xCT, the active subunit of xc⁻, is highly expressed in CRC cell lines and human colorectal carcinoma tissues compared with their normal counterparts. SSZ effectively depleted cellular GSH, leading to significant accumulation of reactive oxygen species and growth inhibition in CRC cells. In contrast, the normal epithelial cells of colon origin were less sensitive to SSZ, showing a moderate ROS elevation. Importantly, SSZ effectively enhanced the intracellular platinum level and cytotoxicity of CDDP in CRC cells. The synergistic effect of SSZ and CDDP was reversed by antioxidant N-acetyl-L-cysteine (NAC). Together, these results suggest that SSZ, a relatively non-toxic drug that targets cystine transporter, may, in combination with CDDP, have effective therapy for colorectal cancer.

Sulfasalazine-induced cystine starvation: Potential use for prostate cancer therapy

Certain cancers depend for growth on uptake of cystine/cysteine from their environment. Here we examined advanced human prostate cancer cell lines, DU-145 and PC-3, for dependence on extracellular cystine and sensitivity to sulfasalazine (SASP), a potent inhibitor of the x(c)(-) cystine transporter.
Cultures were evaluated for growth dependence on exogenous cystine, x(c)(-) transporter expression, response to SASP (growth and glutathione content). In vivo, effect of SASP was determined on subrenal capsule xenograft growth. Cystine omission from culture medium arrested DU-145 and PC-3 cell proliferation; both cell lines expressed the x(c)(-) transporter and were growth inhibited by SASP (IC(50)s: 0.20 and 0.28 mM, respectively). SASP-induced growth inhibition was associated with vast reductions in cellular glutathione content – both effects based on cystine starvation. SASP (i.p.) markedly inhibited growth of DU-145 and PC-3 xenografts without major toxicity to hosts.
SASP-induced cystine/cysteine starvation leading to glutathione depletion may be useful for therapy of prostate cancers dependent on extracellular cystine.

TWO COMMON MEDICINES FOUND TO FIGHT DEADLY CHILDHOOD CANCER

A combination of two medicines long used for treating other illnesses can stop the growth of a deadly childhood cancer, according to a recent study by a Michigan State University College of Human Medicine researcher who has a history of finding new uses for old drugs.

Laboratory tests on cell cultures showed that the drugs DFMO and sulfasalazine effectively impeded the growth of neuroblastoma, which causes about 15 percent of all childhood cancer deaths.

For years DFMO, or difluoromethylornithine, has been used for treating African sleeping sickness, and sulfasalazine for bowel disorders and rheumatoid arthritis.

Others

http://www.bccrc.ca/dept/et/personnel%20-%20content/research-laboratory-technical-stafff/dr.-peter-w.-gout-ph.d
More about how Sulfasalazine may interact with other targets in the body Ref

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