Association between nitric oxide and cancer and stroke risk: A meta-analysis

Introduction

Nitric oxide, or NO, is a chemical compound found in organisms such as mammals. For example, in humans, NO acts as a signaling molecule in various physiological and pathological processes in the body and simultaneously, becomes a diatomic free radical, produced by the enzymatic activity of NOS itself on the L-Arginine compound, yielding the production of L-citrulline along with NO (Korde Choudhari et al., 2013). The NOS family consists of three members, namely eNOS, nNOS, and finally iNOS (Sachdev, 1999). NO influences the coagulation process, neuronal activity, and cerebral blood flow (Korde Choudhari et al., 2013). NO has the potential to induce cellular inflammation, which can delay the onset of stroke. Additionally, NO can act as a carcinogen, increasing cancer risk.

NO plays a substantial role in cancer’s progression and development. NO promotes cancer progression and metastasis via polyamine synthesis or inhibition of NO-mediated tumor cytotoxicity (Gào and Schöttker, 2017; Gào et al., 2019). The roles and functions of NO have been extensively investigated in numerous types of cancer. The response to hypoxia involves NO, which plays a crucial role in inducing angiogenesis and promoting cancer cell defense, and is attributed to the mutagenic behavior exhibited by NO. When cells are exposed to NO for a considerable duration, it is commonly a result of iNOS being produced during chronic inflammation, which gives it a role in carcinogenesis (Utispan and Koontongkaew, 2020). iNOS is considered to have an impact on the mechanism of carcinogenesis.

iNOS plays a multifaceted role in tumor building through its involvement in genetic changes, angiogenesis, proliferation, metastasis, and immunosuppression (Erlandsson et al., 2018). Several studies have proven NO’s influence on both illnesses. NO has been demonstrated to contribute to lung cancer’s progression (Chen et al., 2008). NO can potentially promote the progression of pulmonary carcinoma through a process called protein nitration. NO can also cause head-neck cancer in smokers and people with alcohol use disorders (Patel et al., 2009). The subtype NO, which is iNOS/NOS2, is considered to be correlated with a raised risk of development of prostate cancer (Aaltoma, Lipponen and Kosma, 2001). NO can also result in the onset of breast cancer (Loibl et al., 2002).

NO has a critical part in regulating cerebral circulation and modulating neuronal activity. Microvascular endothelial cells in the brain, also known as the endothelium, are capable of producing and releasing various vasoactive substances, among which NO. The continuous production of NO by the endothelium in basal situations and its reactions to vasoactive stimuli provide knowledge of the complex regulation of cerebral circulation and the maintenance of vascular health in the brain. This dysfunction in NO production and release could be a factor in the progression of stroke. Stroke refers to a clinical condition characterized by unexpected loss of cerebral responsibility as a result of vascular pathology in the brain (Demaerschalk et al., 2016). NO is essential to stroke as an important signaling molecule. The harmful effects of NO derived from iNOS and nNOS primarily stem from the generation of nitrates and free radicals (Zhao et al., 2000). nNOS and iNOS are involved in causing nerve injury during both the beginning and final stages of brain ischemia. Conversely, when eNOS is activated, it has a neuroprotective effect (Chen et al., 2017).

The NOS isoform responsible for producing NO in the vascular endothelium is known as endothelial NOS (eNOS), which in its isoform is expressed through cells and actively contributes to normal vascular tone in physiological conditions. eNOS has also been extensively investigated in the context of carcinogenesis, particularly its involvement in mediating tumor maintenance (Lim et al., 2008). Furthermore, limited levels of NO produced by eNOS can have a neuroprotective effect on stroke through increased vasodilation and cerebral circulation (Yang et al., 2019). Recently, several single nucleotide polymorphisms have been found in eNOS, among which 894G>T in exon 7.

Several case-control studies aimed to check whether the NO correlation with cancer and stroke risk exists, especially its polymorphism eNOS, as well as how these factors may impact the development of cancer and stroke risk. Hence, we conducted a meta-analysis to provide a clearer understanding of the association between NO levels and both cancer risks.

Methods

Results

Study processing

The flow diagram in Figure 1 summarises the study workflow. A total of 145 articles were identified in the databases. After removing duplicates, a total of 105 studies remained. These studies were further screened by reviewing the titles and abstracts, leading to the exclusion of 56 articles that did not meet the predetermined exclusion and inclusion conditions. After carefully examining the full text of the remaining 43 records, an additional 21 articles were excluded based on both exclusion and inclusion conditions. Ten other studies (da Costa Escobar Piccoli et al., 2012; Jang et al., 2013; Rah et al., 2013; Akhter et al., 2014; Kang et al., 2014; Özçelik et al., 2014; Ben Chaaben et al., 2015; Hung et al., 2019; Lee, 2019; Tsay et al., 2019) were also excluded because of unavailable genotype frequency. Six studies (Hao, Montiel and Huang, 2010; Yao et al., 2013; Guo, 2014; Zhao et al., 2014; Abedinzadeh et al., 2020; Akbar et al., 2022) were not included in the analysis because they were meta-analysis studies.

Figure 1. Flow diagram depicting the inclusion process of the studies in the meta-analysis.

Finally, 15 qualified articles met the eligibility criteria. Nine case-control studies examined the association between NO and cancer risk, and six case-control studies analyzed the association between NO and stroke risk. Table 1 shows the features of the 15 studies incorporated in our analysis.

Table 1. Characteristics and genetic frequencies derived the studies included in the meta-analysis.

First Author/Year	Country	Risk	Case/Control	Case					Control
				Genotype			Allele		Genotype			Allele
				GG	TT	GT	T	G	GG	TT	GT	T	G
(Adibmanesh et al., 2020)	Iran	Cancer	100/100	28	28	44	100	100	57	6	37	49	151
(Aouf et al., 2019)	Tunisia	Cancer	259/169	149	20	90	130	388	73	18	78	114	224
(Branković et al., 2013)	Serbia	Cancer	150/100	76	9	65	83	217	54	6	40	52	148
(Carkic et al., 2020)	Serbia	Cancer	50/110	21	5	24	34	66	61	7	42	56	164
(Koçer et al., 2020)	Turkey	Cancer	107/100	74	1	32	34	180	65	1	34	36	164
(Su et al., 2018)	Taiwan	Cancer	1044/1200	825	10	209	229	1859	935	15	250	280	2120
(Verim et al., 2013)	Turkey	Cancer	66/88	7	10	49	69	63	31	13	44	70	106
(Yadav et al., 2019)	India	Cancer	179/173	88	20	64	104	240	96	8	59	75	251
(Yanar et al., 2016)	Turkey	Cancer	58/147	18	11	29	51	65	31	35	81	151	143
(Anliaçik et al., 2019)	Turkey	Stroke	112/160	21	14	77	44	40	38	19	103	57	61
(Diakite et al., 2014)	Morocco	Stroke	165/182	83	16	66	30	70	117	7	58	20	81
(El Gohary, El Azab and Kamal El-Din, 2017)	Egypt	Stroke	30/10	18	6	6	45	15	5	3	3	12	8
(Kaur, Uppal and Kaur, 2015)	India	Stroke	120/101	84	6	30	18	83	83	1	17	9	91
(Kumar et al., 2016)	India	Stroke	250/250	164	12	74	20	80	186	5	59	14	86
(Shyu et al., 2017)	Taiwan	Stroke	229/243	151	16	62	21	50	185	7	51	13	87

Heterogeneity among studies

Heterogeneity was observed among the studies in every allele and gene, as depicted in Figures 2 and 3 (T versus G, G versus T, GT+TT versus GG, TT versus GG + GT, and GT versus GG + TT). Tables 2 and 3 provide information on the selected model (random or fixed effect) utilized in order to review universal genetic model correlations.

Figure 2. Forest plots depicting the association of eNOS 894G>T polymorphisms with cancer risk in all genetic models, including T vs G, G vs T, GT + TT vs GG, TT vs GG + GT, and GT vs GG + TT, respectively.

Figure 3. Forest plots illustrating the association of eNOS 894G>T polymorphisms with stroke risk in all genetic models, including T vs G, G vs T, GT + TT vs GG, TT vs GG + GT, and GT vs GG + TT, respectively.

Discussion

NO acts as a crucial part of numerous pathological and psychological processes. NO is extensively implicated in various events related to cancer, including angiogenesis, metastasis, invasion, and apoptosis, which various studies have investigated; results have provided evidence that increased concentrations of NO within cancer cells can effectively suppress tumor angiogenesis and metastasis (Zhao et al., 2014). Conversely, low levels of NO in tumor cells may facilitate tumor growth by reducing NO-induced apoptosis (Heller, 2008). These observations suggest that the effects of NO on cancer development may be strongly dependent on the local NO concentration. NO is the result of three types of NOS isoforms, namely nNOS, eNOS, and iNOS. These enzymes facilitate the conversion of l-arginine to l-citrulline through oxidation (Vanini, Kashfi and Nath, 2015). eNOS is one of the three isoforms of NOS responsible for synthesizing NO in humans. Moreover, this particular isoform is closely linked to angiogenesis, which is associated with NO synthesis in both normal and cancerous cells (Song et al., 2013). A polymorphism was found in the gene encoding eNOS that could alter the production of NO. The G894T variation, also known as the guanine polymorphism to thymine with position 894 and exon 7 (rs1799983), is of particular interest (Akbar et al., 2022).

From several studies that were selected for our meta-analysis, the polymorphism was correlated with increased cancer risk in both African, European, and Asian countries. Adibmanesh et al. (2020) stated that the eNOS polymorphism 894G>T showed a significant correlation with colorectal cancer existing in the T allele genotype. Aouf et al. (2019) stated that NOS3 G894T substantially increases the risk of nasopharyngeal carcinoma (NPC) in a population from Tunisia. Furthermore, research conducted by Branković et al. (2013) suggested that NOS3 894G>T genetic polymorphisms were not associated with the risk of prostate tumor in a community in Serbia but may be relevant as prognostic factors for the progression of prostate cancer and patients’ outcome. In the study by Carkic et al. (2020), the results proved that eNOS had a significant impact on oral squamous cell cancer (OSCC) in the Serbian population. Koçer et al. (2020) stated that there was no significant association between the eNOS G894T gene and the risk of lung cancer, where p was greater than 0.05. Research by Su et al. (2018) found no relationship between OSCC and eNOS holotypes in Taiwan. Research by Verim et al. (2013) proved the existence of NOS3 is crucial in increasing the susceptibility to bladder cancer within the Turkish population. Based on the research by Yadav et al. (2019), it is suggested that eNOS 894G > T polymorphisms play a role in influencing the risk of epidermoid cell cancer of the head and neck in the population of North India. Additionally, a study conducted by Yanar et al. (2016) suggests a potential association between the G894T variation of NOS3 and the possibility of laryngeal cancer (LC), possibly due to the involvement of impaired redox homeostasis.

The role of nitric oxide in cancer can be seen in Figure 4. Based on Figure 4, overproduction of NO can facilitate tumor angiogenesis and metastasis. The NOS isoforms that produce NO in the vascular endothelium are defined as endothelial NOS (eNOS), which is found in the endothelium and carries out a crucial role in regulating vascular tone under normal conditions, which is involved in carcinogenesis and contributes to tumor protection (Lim et al., 2008). One possible explanation for the role of this enzyme in cancer progression is that reduced eNOS enzyme activity may lead to a functional decrease in NO levels within the tumor microenvironment, thereby promoting tumor growth. Recently, single nucleotide polymorphisms (SNPs) have been discovered in the eNOS gene. One of these SNPs, located at exon 7 (894G>T). Regarding to the functional role of NO in regulating angiogenesis in cancer, it is possible that this SNP might be positively correlated with the cancer progress by affecting NO synthesis.

Figure 4. The role of nitric oxide in cancer.

Stroke ranks as the second-leading major contributor to mortality and disability in adults, after coronary heart disease (WHO, 2020). Stroke is a multifactorial disease; Epidemiological studies and animal experiments have provided indications of a genetic impact on the development of ischemic stroke (IS) (Hassan and Markus, 2000). Family history also serves as a crucial factor in assessing the potential for stroke. Endothelial NO, synthesized by eNOS, acts as a significant part of regulating blood flow and exhibits anti-proliferation and anti-inflammatory substances. eNOS polymorphism has a significant impact on endothelial dysfunction. The G894T variant of eNOS has been implicated in the development of diverse conditions, consisting of cardiovascular diseases and erectile dysfunction. A compromised NO-dependent vasomotor response is believed to be involved in the pathophysiology of stroke (Kaur, Uppal and Kaur, 2015). Because of its significant role in vascular physiology, genetic mutations may contribute to stroke pathogenesis by altering the expression and enzymatic activity of eNOS.

External influences that affect eNOS cause cancer and stroke through chronic stress. eNOS activity is regulated by adrenaline (Seya et al., 2006; Kou and Michel, 2007; Figueroa et al., 2009; Barbieri et al., 2012). Prolonged stress can act as a contributing factor in the onset and advancement of cancer. Stress is also considered a relevant factor in cancer development (Antoni et al., 1978; Chida et al., 2008; Desaive and Ronson, 2008). eNOS plays an essential role in ensuring vascular homeostasis, which includes regulating vascular integrity, blood flow, cell adhesion, angiogenesis, vascular permeability, immune response, and metabolism. Additionally, chronic stress can elevate the production of specific growth factors that enhance blood supply (Heid, 2014). This can accelerate the progression of cancerous tumors. Furthermore, stress can lead to increased cardiac burden, elevated blood pressure, and raised levels of sugar and fat in the bloodstream (Heart and Stroke, 2023). These factors can elevate the risk of cerebral blood clot formation, resulting in increased susceptibility to stroke.

From for the studies examined for the present meta-analysis, the presence of the eNOS polymorphism 894G>T has been correlated with a raised susceptibility to stroke in individuals of African, Asian, and European ancestry. Research conducted by Anliaçik et al. (2019) indicates there is no significant relationship between eNOS G894T and ischemia stroke among the Anatolia population. Diakite et al. (2014) stated that a significant relationship has been observed between the eNOS polymorphism 894G>T and ischemia stroke found in dominant, recessive, and additive models in the Moroccan population. Furthermore, research conducted by El Gohary, El Azab, and Kamal El-Din (2017) stated that no significant association was found between the eNOS polymorphism G894T and immediate stroke in Egyptian patients.

Research conducted by Kaur, Uppal, and Kaur (2015) stated that the G894T variant has been found to be associated with ischemic stroke and may contribute to ischemic stroke susceptibility in the Northern Indian population Kumar et al. (2016) suggest that the G894T eNOS can be a determinant of ischemic stroke, mainly for the large vessel disease (LVD) subtype, in the Northern Indian population. Additionally, a study by Shyu et al. (2017) reported that genotypic polymorphisms of the eNOS G894T polymorphism were given or used as an optimization of the risk of atherosclerotic stroke in Taiwan.

The role of nitric oxide in stroke can be seen in Figure 5. Based on Figure 5, low levels of NO derived from eNOS may exert neuroprotection in stroke by promoting vasodilatation and increasing cerebral blood flow (Yang et al., 2019). However, at the same time, there is an enhancement in superoxide production due to eNOS uncoupling. This leads to a significant increase in peroxynitrite formation, which damages lipids, proteins, and DNA and can trigger activation of poly adenosine diphosphate rribose (ADP-ribose) polymerase (PARP), which all contribute to neurotoxicity in stroke.

Figure 5. The role of nitric oxide in stroke.

We conducted a meta-analysis considering the association or correlation of nitric oxide, especially NOS 894G>T polymorphism with stroke, and cancer risk. A total of 15 studies included 3,333 controls and 3,019 cases were selected for this meta-analysis. Overall, eNOS polymorphism 894G>T was found to be significantly correlated with increased cancer risk in the GG versus GT+TT genetic model. However, there were no substantial associations in the genetic model as examined in G versus T, T versus G, GT versus GG+TT, and TT versus GG+GT. The polymorphism has a relationship with a substantially higher risk of stroke in the genetic models of both TT versus GG+GT and T versus G.

Conclusions

In conclusion, the recent meta-analysis found that nitric oxide-related polymorphisms with the eNOS 894G>T gene are associated with a substantial risk of cancer in the total population based on the GG vs. GT+TT genetic model and significantly correlated with the manifestation of stroke in the genetic models T vs. G, and TT vs. GG + GT. G vs. T, and GG + GT vs. TT. Considering the conclusion, these results should be reassessed in the coming days through studies with a larger sample population.