Posts Tagged ‘pro-apoptotic’

Reporter/Curator: Stephen J. Williams, Ph.D.

Picture of a human melanoma cell line growing in tissue culture

Cultured human melanocytes .

Nitric oxide (NO), a gas with many biological functions in healthy cells, has also been implicated in the development of pathologies such as cancer.  Nitric oxide may also play a role in chemotherapeutic reisitance. For example it had been known (in the 1996 Melanoma study by Joshi et al. curated below) that nitric oxide synthase activity (the enzyme system which produces NO) was significantly elevated in cultured melanoma cell lines versus normal melanocytes.   Although it is known that many protein and enzymes systems could be directly covalently-modified by nitric oxide, either by S-nitrosylation or NO-NAD+ modifications (one of my earlier postings described one such protein modified by nitric oxide, GAPDH, and the effect these NO-modifications of GAPDH has on the etiology of various pathologies.), the molecular mechanisms by which these modifications affect cellular processes, lead to disease etiology, the proteins which are affected, and mechanisms related to chemotherapeutic sensitivity need to be further characterized. A new study from MIT reveals how NO-induced modifications may reduce cisplatin sensitivity in melanoma cells.  This study focuses on how decreasing nitric oxide levels in melanoma cells increases their cisplatin sensitivity.  The study also describes a possible mechanism for this effect: NO-induced modifications of the proapoptotic enzyme caspace-3 and prolyl-hdroxylase-2 (responsible for targeting prosurvival HIF-1α for proteosomal degradation).  Also, for a description of other cancer-related targets of nitric oxide please see the posting by Dr. Saxena at Crucial role of Nitric Oxide in Cancer on this site.

To read more background on nitric oxide and its role in disease etiology please see our e-book Perspectives on Nitric Oxide in Disease Mechanisms (Biomed e-Books) available on Amazon at:

      It is important, however, to note that most of these relationships between NO-induced protein modification and its relationship to disease mechanisms are causal, meaning that, in general, one notices a nitric-induced modification of a protein/enzyme with concomitant alteration of protein/enzyme function occurring in a disease/phenotype.  However, unlike reversible modifications, which have a cadre of pharmacologic inhibitors, nitric oxide induced modifications are covalent and nonenzymatic, therefore hindering easy cause/effect relationships.

With that said, the following was adapted from the MIT site at



The findings from Dr. Luiz Godoy’s PNAS paper ENDOGENOUSLY PRODUCED NITRIC OXIDE MITIGATES SENSITIVITY OF MELANOMA CELLS TO CISPLATIN,  were presented at the 2013 annual meeting of the American Association for Cancer Research. The prognosis is generally worse for patients whose tumors have high levels of NO, said Luiz Godoy, an MIT research associate and lead author of the study.

Godoy and his colleagues have unraveled the mechanism behind melanoma’s resistance to cisplatin, a commonly used chemotherapy drug, and, in ongoing studies, have found that cisplatin treatment also increases NO levels in breast and colon cancers.

“This could be a mechanism that is widely shared in different cancers, and if you use the drugs that are already used to treat cancer, along with other drugs that could scavenge or decrease the production of NO, you may have a synergistic effect,” said Godoy, who works in the lab of Gerald Wogan, an MIT professor emeritus of biological engineering and senior author of the study.

NO has many roles within living cells. At low concentrations, it helps regulate processes such as cell death and muscle contraction. NO, which is a free radical, is also important for immune-system function. Immune cells, such as macrophages, produce large amounts of NO during infection, helping to kill invading microbes by damaging their DNA or other cell components.

“It’s really a molecule that has a dual effect,” Godoy said. “At low concentrations it can act as a signaling molecule, while high concentrations will be toxic.”

Knocking out NO

In the new study, the researchers treated melanoma cells grown in the lab with drugs that capture NO before it can act. They then treated the cells with cisplatin and tracked cell-death rates. The NO-depleted cells became much more sensitive to the drug, confirming earlier findings.

The MIT team then went a step further, investigating how NO confers its survival benefits. It was already known that NO can alter protein function through a process known as S-nitrosation, which involves attaching NO to the target protein. S-nitrosation can affect many proteins, but in this study the researchers focused on two that are strongly linked with cell death and survival, known as caspase-3 and PHD2.

The role of caspase-3 is to stimulate cell suicide, under the appropriate conditions, but adding NO to the protein deactivates it. This prevents the cell from dying even when treated with cisplatin, a drug that produces massive DNA damage.

PHD2 is also involved in cell death; its role is to help break down another protein called HIF-1 alpha, which is a pro-survival protein. When NO inactivates PHD2, HIF-1 alpha stays intact and keeps the cell alive.

“Now we have a mechanistic link between nitric oxide and the increased aggressiveness of melanoma,” said Douglas Thomas, an assistant professor of medicinal chemistry and pharmacognosy at the University of Illinois at Chicago, who was not part of the research team. “It certainly would be worth exploring whether this mechanism is also present in different tumor types as well.”

The MIT researchers also found in some cancer cells, NO levels were five times higher than normal following cisplatin treatment. Godoy is now investigating how cisplatin stimulates that NO boost, and is also looking for other proteins that NO may be targeting.


Melanoma Res. 1996 Apr;6(2):121-6.

Nitric oxide synthase activity is up-regulated in melanoma cell lines: a potential mechanism for metastases formation.

Joshi M, Strandhoy J, White WL.


Department of Dermatology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA.


Nitric oxide (NO) may be an important mediator of tumour angiogenesis and metastasis formation. Tumour cell derived NO may be important in the regulation of angiogenesis and vasodilatation of the blood vessels surrounding a tumour. The aims of the present study were, firstly, to determine whether malignant melanoma cells and normal melanocytes had nitric oxide synthase (NOS) activity (measured by the conversion of L-arginine to L-citrulline) and, secondly, to determine whether there was a difference in NOS activity between malignant and normal cell types. This paper assays NOS activity directly in lysates from normal human melanocyte and malignant melanoma cell lines. The enzyme activity was not inducible with bacterial lipopolysaccharide and could be heat denatured. The activity of NOS was demonstrated to be both NADPH- and calcium-dependent and it was inhibitable in a dose-dependent manner by the NOS inhibitor Nw-nitro-L-arginine methyl ester. We conclude that melanoma and melanocyte cells express a constitutive form of NOS. Finally, nitric oxide synthase activity in melanoma cell lines was found to be significantly greater than in normal melanocytes. These findings suggest that NO synthesis is elevated in malignant melanoma. An elevated NO concentration in melanoma is expected to promote metastases by maintaining a vasodilator tone in the blood vessels in and around the melanoma.

Proc Natl Acad Sci U S A. 2012 Dec 11;109(50):20373-8. doi: 10.1073/pnas.1218938109. Epub 2012 Nov 26.

Endogenously produced nitric oxide mitigates sensitivity of melanoma cells to cisplatin.

Godoy LC, Anderson CT, Chowdhury R, Trudel LJ, Wogan GN.


Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.


Melanoma patients experience inferior survival after biochemotherapy when their tumors contain numerous cells expressing the inducible isoform of NO synthase (iNOS) and elevated levels of nitrotyrosine, a product derived from NO. Although several lines of evidence suggest that NO promotes tumor growth and increases resistance to chemotherapy, it is unclear how it shapes these outcomes. Here we demonstrate that modulation of NO-mediated S-nitrosation of cellular proteins is strongly associated with the pattern of response to the anticancer agent cisplatin in human melanoma cells in vitro. Cells were shown to express iNOS constitutively, and to generate sustained nanomolar levels of NO intracellularly. Inhibition of NO synthesis or scavenging of NO enhanced cisplatin-induced apoptotic cell death. Additionally, pharmacologic agents disrupting S-nitrosation markedly increased cisplatin toxicity, whereas treatments favoring stabilization of S-nitrosothiols (SNOs) decreased its cytotoxic potency. Activity of the proapoptotic enzyme caspase-3 was higher in cells treated with a combination of cisplatin and chemicals that decreased NO/SNOs, whereas lower activity resulted from cisplatin combined with stabilization of SNOs. Constitutive protein S-nitrosation in cells was detected by analysis with biotin switch and reduction/chemiluminescence techniques. Moreover, intracellular NO concentration increased significantly in cells that survived cisplatin treatment, resulting in augmented S-nitrosation of caspase-3 and prolyl-hydroxylase-2, the enzyme responsible for targeting the prosurvival transcription factor hypoxia-inducible factor-1α for proteasomal degradation. Because activities of these enzymes are inhibited by S-nitrosation, our data thus indicate that modulation of intrinsic intracellular NO levels substantially affects cisplatin toxicity in melanoma cells. The underlying mechanisms may thus represent potential targets for adjuvant strategies to improve the efficacy of chemotherapy.

Other posts on this site regarding Nitric Oxide and Cancer include:

Crucial role of Nitric Oxide in Cancer

Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Nitric Oxide Signalling Pathways

In focus: Melanoma therapeutics

Combined anti-CTLA4 and anti-PD1 immunotherapy shows promising results against advanced melanoma

Whole exome somatic mutations analysis of malignant melanoma contributes to the development of personalized cancer therapy for this disease

In focus: Melanoma therapeutics

In focus: Melanoma Genetics


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Author and Curator: Ritu Saxena, Ph.D.



Nitric oxide (NO) is a lipophilic, highly diffusible and short-lived molecule that acts as a physiological messenger and has been known to regulate a variety of important physiological responses including vasodilation, respiration, cell migration, immune response and apoptosis. Jordi Muntané et al

NO is synthesized by the Nitric Oxide synthase (NOS) enzyme and the enzyme is encoded in three different forms in mammals: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3). The three isoforms, although similar in structure and catalytic function, differ in the way their activity and synthesis in controlled inside a cell. NOS-2, for example is induced in response to inflammatory stimuli, while NOS-1 and NOS-3 are constitutively expressed.

Regulation by Nitric oxide

NO is a versatile signaling molecule and the net effect of NO on gene regulation is variable and ranges from activation to inhibition of transcription.

The intracellular localization is relevant for the activity of NOS. Infact, NOSs are subject to specific targeting to subcellular compartments (plasma membrane, Golgi, cytosol, nucleus and mitochondria) and that this trafficking is crucial for NO production and specific post-translational modifications of target proteins.

Role of Nitric oxide in Cancer

One in four cases of cancer worldwide are a result of chronic inflammation. An inflammatory response causes high levels of activated macrophages. Macrophage activation, in turn, leads to the induction of iNOS gene that results in the generation of large amount of NO. The expression of iNOS induced by inflammatory stimuli coupled with the constitutive expression of nNOS and eNOS may contribute to increased cancer risk. NO can have varied roles in the tumor environment influencing DNA repair, cell cycle, and apoptosis. It can result in antagonistic actions including DNA damage and protection from cytotoxicity, inhibiting and stimulation cell proliferation, and being both anti-apoptotic and pro-apoptotic. Genotoxicity due to high levels of NO could be through direct modification of DNA (nitrosative deamination of nucleic acid bases, transition and/or transversion of nucleic acids, alkylation and DNA strand breakage) and inhibition of DNA repair enzymes (such as alkyltransferase and DNA ligase) through direct or indirect mechanisms. The Multiple actions of NO are probably the result of its chemical (post-translational modifications) and biological heterogeneity (cellular production, consumption and responses). Post-translational modifications of proteins by nitration, nitrosation, phosphorylation, acetylation or polyADP-ribosylation could lead to an increase in the cancer risk. This process can drive carcinogenesis by altering targets and pathways that are crucial for cancer progression much faster than would otherwise occur in healthy tissue.

NO can have several effects even within the tumor microenvironment where it could originate from several cell types including cancer cells, host cells, tumor endothelial cells. Tumor-derived NO could have several functional roles. It can affect cancer progression by augmenting cancer cell proliferation and invasiveness. Infact, it has been proposed that NO promotes tumor growth by regulating blood flow and maintaining the vasodilated tumor microenvironment. NO can stimulate angiogenesis and can also promote metastasis by increasing vascular permeability and upregulating matrix metalloproteinases (MMPs). MMPs have been associated with several functions including cell proliferation, migration, adhesion, differentiation, angiogenesis and so on. Recently, it was reported that metastatic tumor-released NO might impair the immune system, which enables them to escape the immunosurveillance mechanism of cells. Molecular regulation of tumour angiogenesis by nitric oxide.

S-nitrosylation and Cancer

The most prominent and recognized NO reaction with thiols groups of cysteine residues is called S-nitrosylation or S-nitrosation, which leads to the formation of more stable nitrosothiols. High concentrations of intracellular NO can result in high concentrations of S-nitrosylated proteins and dysregulated S-nitrosylation has been implicated in cancer. Oxidative and nitrosative stress is sensed and closely associated with transcriptional regulation of multiple target genes.

Following are a few proteins that are modified via NO and modification of these proteins, in turn, has been known to play direct or indirect roles in cancer.

NO mediated aberrant proteins in Cancer


Bcl-2 is an important anti-apoptotic protein. It works by inhibiting mitochondrial Cytochrome C that is released in response to apoptotic stimuli. In a variety of tumors, Bcl-2 has been shown to be upregulated, and it has additionally been implicated with cancer chemo-resistance through dysregulation of apoptosis. NO exposure causes S-nitrosylation at the two cysteine residues – Cys158 and Cys229 that prevents ubiquitin-proteasomal pathway mediated degradation of the protein. Once prevented from degradation, the protein attenuates its anti-apoptotic effects in cancer progression. The S-nitrosylation based modification of Bcl-2 has been observed to be relevant in drug treatment studies (for eg. Cisplatin). Thus, the impairment of S-nitrosylated Bcl-2 proteins might serve as an effective therapeutic target to decrease cancer-drug resistance.


p53 has been well documented as a tumor suppressor protein and acts as a major player in response to DNA damage and other genomic alterations within the cell. The activation of p53 can lead to cell cycle arrest and DNA repair, however, in case of irrepairable DNA damage, p53 can lead to apoptosis. Nuclear p53 accumulation has been related to NO-mediated anti-tumoral properties. High concentration of NO has been found to cause conformational changes in p53 resulting in biological dysfunction.. In RAW264.7, a murine macrophage cell line, NO donors induce p53 accumulation and apoptosis through JNK-1/2.


Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that is predominantly active under hypoxic conditions because the HIF-1a subunit is rapidly degraded in normoxic conditions by proteasomal degradation. It regulates the transciption of several genes including those involved in angiogenesis, cell cycle, cell metabolism, and apoptosis. Hypoxic conditions within the tumor can lead to overexpression of HIF-1a. Similar to hypoxia-mediated stress, nitrosative stress can stabilize HIF-1a. NO derivatives have also been shown to participate in hypoxia signaling. Resistance to radiotherapy has been traced back to NO-mediated HIF-1a in solid tumors in some cases.


Phosphatase and tensin homolog deleted on chromosome ten (PTEN), is again a tumor suppressor protein. It is a phosphatase and has been implicated in many human cancers. PTEN is a crucial negative regulator of PI3K/Akt signaling pathway. Over-activation of PI3K/Akt mediated signaling pathway is known to play a major role in tumorigenesis and angiogenesis. S-nitrosylation of PTEN, that could be a result of NO stress, inhibits PTEN. Inhibition of PTEN phosphatase activity, in turn, leads to promotion of angiogenesis.


C-src belongs to the Src family of protein tyrosine kinases and has been implicated in the promotion of cancer cell invasion and metastasis. It was demonstrated that S-nitrosylation of c-Src at cysteine 498 enhanced its kinase activity, thus, resulting in the enhancement of cancer cell invasion and metastasis.


Muntané J and la Mata MD. Nitric oxide and cancer. World J Hepatol. 2010 Sep 27;2(9):337-44.

Wang Z. Protein S-nitrosylation and cancer. Cancer Lett. 2012 Jul 28;320(2):123-9.

Ziche M and Morbidelli L. Molecular regulation of tumour angiogenesis by nitric oxide. Eur Cytokine Netw. 2009 Dec;20(4):164-70.

Jaiswal M, et al. Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis. Am J Physiol Gastrointest Liver Physiol. 2001 Sep;281(3):G626-34.

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