Effect of Cigarette versus Electronic Cigarette on the Expression of TERT, FGFR3, PTEN, P53, and VEGF in Rat Bladder

Introduction

Bladder cancer is the 10th most common cancer worldwide and could be categorized as non-muscle-invasive and muscle-invasive bladder cancer. In 2018, approximately more than half a million new bladder cancer cases were diagnosed globally, and 200,000 died from this cancer (1). The cigarette is the leading risk factor for bladder cancer, accounting for approximately 50–65% of new cases each year. Smoking has been shown to raise the chance of bladder cancer by three to four times (2). Recently, significant smokers have switched to electronic cigarettes (e-cigarettes), and approximately 3.6 million youths use e-cigarettes (3).  E-cigarettes are aggressively advertised as smoking cessation or reduction aid and a cheaper, more ecological, socially acceptable, and healthier alternative to conventional cigarettes. Numerous studies have assessed the effects of cigarette chemicals on genetic and epigenetic pathways that contribute to inflammation or change the cell cycle (4),  while the effect of e-cigarettes on these pathways is unclear.

Several novel molecular technologies are considered to detect diagnostic biomarkers of bladder tumors (5, 6). Mutations drive bladder cancer in different sets of genetic pathways. Studies indicated the high rate of telomerase reverse transcriptase (TERT) and fibroblast growth factor receptor 3 (FGFR3) promotor mutations in bladder cancer that play critical roles in the tumorigenesis and pathogenesis of bladder cancer (5, 7-9). TERT mutation is the most frequent somatic genetic alteration in non-muscle-invasive and muscle-invasive urothelial cell carcinoma. Moreover, FGFR3 mutation is a common genetic event in non-muscle-invasive bladder neoplasm (10). Also, the vascular endothelial growth factor (VEGF) gene plays a crucial role in angiogenesis and tumor growth. VEGF gene polymorphisms and bladder cancer risk have been studied extensively (11). On the other hand, tumor suppressor genes, including P53 and PTEN, are the most frequently involved gene in cancer in human beings and have been the most widely investigated in bladder cancer in the past decade (12, 13).

Previous studies assessed the effect of cigarettes and their main ingredients on the mutation of critical genes involved in the development of bladder cancer. Still, very few studies investigated the impact of smoking on the expression of these genes. On the other hand, the pattern of smoking has changed, and most smokers switch to e-cigarettes, and the impact of e-cigarettes is not clear on the expression of these genes. According to our knowledge, no study evaluated and compared the effects of cigarettes and e-cigarettes on the expression of critical genes involved in bladder cancer. Therefore, the present study aimed to assess and compare the impact of cigarettes and e-cigarettes on the expression of central genes involved in the development of bladder cancer.

Method

This experimental study was performed in the Animal Laboratory of Urology Research Center of Tehran Medical University, Tehran, Iran, from September 2021 to April 2022 and was approved by the Ethics Research Committee of Tehran University of Medical Sciences (IR.TUMS.SINAHOSPITAL.REC.1399.019). Ethical principles of working with animals were considered in all stages of the research.

Animal Caring

60 Wistar rats, 30 male rats, and 30 female rats (6 weeks, weighing 218 ± 22 g), ten rats in each group, from Pasteur Institute, Tehran, Iran (standard place for breeding laboratory animals) were prepared. The rats were kept in six clean plastic cages (18 x 22 x 30 cm), cleaned, and disinfected every two days. All rats were fed special diets containing protein and fat prepared by the Royan Institute, Iran, as a commercial feed for laboratory animals, and all rats had free access to tap water. The rats were kept for two weeks before the start of the study at the Urology Research Center Animal laboratory to adapt to the environment and relieve the rats' stress. The room temperature of rats was about (22±2°C) with humidity (55±5%), 12 hours of light, and 12 hours of darkness. At the beginning and end of the study, the weights of the rats were recorded, and the male and female rats were kept separately in cages.

Exposure of Rats to cigarette smoke

Rats were categorized into three groups (n=10):  Group A (Control), Group B: Case-1 (Cigarette), and Group C: Case-2 (E-cigarette). Subsequently, the smoke of cigarettes or e-cigarette enters the plastic cages via a specially designed suction device. The rats were exposed to the smoke of cigarettes and e-cigarette three times a day for 40 minutes with a one-hour interval (1.8 mg nicotine/day) for 16 weeks. Each used cigarette contains 0.6 mg of nicotine, equivalent to 24µL of nicotine liquid from the e-cigarette used in this research. Each month of a mouse's lifespan is approximately 2.5 - 3 human years (14, 15). Therefore, four months of rats' exposure to cigarettes and e-cigarette equals ten to twelve years of smoking in humans.

Investigation of serum parameters and bladder pathology examination

Blood sampling was done before starting the study and at the end of the study. A complete blood count (CBC) was performed with automated hematology and an ESR analyzer (Nihon kohden MEK-1305 Celltac α+). The urinary bladders were removed from the rats and placed in 10% neutral-buffered formalin. The specimens were sent to the comparative pathology laboratory after fixation time. Histological studies were performed in the pathology department of the Tehran Medicine University, Urology Research Center. An automated autotechnicon tissue processor processed the samples. After molding, different slice sections (5-micron thick) were prepared from blocks of embedded tissues by a microtome machine. Then, all slides were stained according to the H & E (hematoxylin and eosin) standard protocol and observed under an Olympus light microscope.

RNA isolation and Real-Time PCR

Total RNA was extracted from bladder tissue by a High Pure RNA Isolation Kit (Cat. No. 11 828 665 001). The quality and quantity of extracted RNA were assessed by Nanodrop ND-1,000 (Technologies, Wilmington, DE). Then, cDNAs were synthesized from the Prime-Script RT reagent Takara Kit (Bio Inc, Otsu, Japan, Cat. #RR014A/B). QIAGEN's real-time PCR cycler calculated the relative expression via the 2−ΔΔCT method. For normalizing the gene expression levels used, B2M is the housekeeping gene. All steps were performed according to the factory instructions. The information on the primers used along with their product lengths is presented in Table 1.

Table 1. Primers used for Real-Time PCR

Statistical analysis

Data were evaluated by Graph Pad prism 9. Two tests, a One-way analysis of variance used for CBC analysis and a t-test used for gene relative expression, were accomplished for statistical analysis. The range of statistical significance was demarcated at * P-value <0.05, ** P-value <0.01, and ***P-value<0.001.

Results

At the end of the study, WBC, MCV, MCH, and RDW increased in male rats in both cigarette and e-cigarette groups. Significantly, PLT increased in both groups, and WBC rose just in the cigarette group (Table 2). In the female rat, PLT increased significantly in both groups, and WBC increased in the e-cigarette group (Table 3).

Table 2. Hematologic parameters in cigarette and e-cigarette groups in male rats

*statistically significant with a range of P-value <0.05.

Table 3. Hematologic parameters in cigarette and e-cigarette groups in female rats

*statistically significant with a range of P-value <0.05.

Histopathological finding

In the case groups compared to the control group, pathological findings based on the proliferation of transitional cells indicate possible tissue cancer. Transitional cell hyperplasia was observed in the cigarette and electronic cigarette groups with an approximate 2: 1 in the two case groups. The growth and proliferation of bladder tissue cells were also observed in female rats of the same groups. The occurrence of vascular reactions, especially inflammation caused by active hyperemia and inflammatory infiltration of lymphocytes, was observed as a pre-neoplastic stage in male rats.

We demonstrated increased transitional cell hyperplasia in female cigarette rats (++) and e-cigarette female rats (+) (in a 2:1 ratio, approximately). Also, we indicated an incidence of hyperemia and inflammatory cell infiltration in e-cigarette male rats (+) (Figures 1 and 2, Table 4).

Figure 1. Histopathological sections of the urinary bladder, female rats, H&E staining. Panel c (× 400) shows a higher magnification of photomicrographs than panel b (× 200), and this panel is higher than panel an (x 100). Stars demonstrate transitional cell hyperplasia.

Figure 2. Histopathological sections of the urinary bladder, male rats, H&E staining. Panel c (× 400) shows a higher magnification of photomicrographs than panel b (× 200), and this panel is higher than panel an (x 100). Arrows demonstrate hyperemia, and arrowheads show lymphocytic cell infiltration.

Table 4. The histopathology examination reports on bladder tissue in both groups of cigarettes and e-cigarettes more than the control group.

The plus signs indicate the presence of parameters (++ indicates a higher incidence than +). While the minus sign indicates the absence of a parameter.

The effect of cigarette smoke on the expression of tumor suppressor gene and angiogenesis pathway 

Through upregulating FGFR3, VEGF, and TERT and downregulating PTEN and P53, e-cigarette smoke might contribute to bladder cancer, although further studies are required to substantiate this proposal. 

FGFR3 gene was significantly increased in both cigarettes (6.82 fold; P-value<0.0001) and e-cigarette groups (2.28 fold; P-value<0.0001) compared to the control group in the male rats. In contrast, these changes were not significant in male rats between the two case groups. Gene expression of FGFR3 was significantly increased in both cigarettes (1.37 fold; P-value<0.0001) and e-cigarette groups (2.71 fold; P-value<0.0001) compared to the control group. In contrast, between the two cigarette and e-cigarette groups, these changes were not significant in female rats. Gene expression of TERT significantly increased in both cigarette and e-cigarette groups in male groups compared to the control no cigarettes exposed group (P-value <0.0001). The same result was observed in female groups but not significantly different.

VEGF significantly increased in both cigarette (2.80 fold; P-value< 0.0001) and e-cigarette (3.30 fold; P-value<0.0001) groups in male rats, and there was a significant difference between the two cigarette and e-cigarette groups (P-value=0.0055). In female rats, the expression of VEGF increased in the e-cigarette group compared to the control (2.12-fold; P-value<0.0001), and there was a significant difference between the cigarette and e-cigarette groups (P-value<0.0001).

P53 expression significantly decreased in female rats in the cigarette group (0.51-fold; P-value=0.0373). Still, in the e-cigarette group, the expression of P53 increased significantly (1.25-fold; P-value=0.0013), and a significant difference was observed between the two cigarette and e-cigarette groups (P-value<0.0001). In the male rat, expression of the P53 decreased significantly in cigarette (0.04-fold; P-value=0.0316) and non-significantly in e-cigarette (0.13-fold; P-value=0.9148) groups, and a significant difference was not found between the two cigarette and e-cigarette groups (P-value=0.1700). 

PTEN was decreased in both cigarette (0.14-fold) and e-cigarette (1.15-fold) groups in male rats compared to the control group, while between the two cigarette and e-cigarette groups, these changes were not significant (P-value >0.05). PTEN was significantly reduced in the female rat in the cigarette group (0.02-fold; P-value=0.0148), while this gene was decreased no significantly in the e-cigarette group (0.151-fold) compared to the control group and between two cigarette and e-cigarette groups (Figures 3 and 4).

Figure 3. Results of case and control groups exposed to cigarettes and e-cigarettes on gene expression (mRNA fold change). Values are given as mean ± SD of three independent experiments.

Figure 4. Results of case and control groups exposed to cigarettes and e-cigarettes on gene expression.Figure 4. Results of case and control groups exposed to cigarettes and e-cigarettes on gene expression.

Discussion

In the present study, the impact of cigarettes and e-cigarettes were evaluated on the expression of primary genes involved in the development of bladder cancer. Overall, the study's findings showed that both types of cigarettes induced hematologic changes, histopathological changes, and gene disturbance in rat bladder. At first, we evaluated the hematologic parameters, and the changes in blood cells approved that the rats were exposed to cigarette smoking and the previous studies showed that the white blood cell increased in smokers (16, 17). Histopathological findings showed cigarette-induced hyperplasia and e-cigarette-induced hyperemia and infiltration of inflammatory cells. Our results were consistent with previous studies (18, 19). The present study's findings showed that both cigarettes and e-cigarettes promoted tumorigenesis by upregulating the expression of VEGF, FGFR3, and TERT and downregulating the expression of tumor suppressor genes, including tumor suppression P53 and PTEN. Consequently, both cigarettes and e-cigarettes might have contributed to the development of bladder cancer, but there were no significant differences between the two types of cigarettes, and both types of cigarettes have the same risk of changing genes related to bladder cancer.

TERT recreates a critical function in cancer formation, providing chromosomal stability by preserving telomere length and permitting cells to prevent senescence (20). This is the first study focused on the effect of cigarettes and e-cigarettes on the TERT expression in rat bladder, and no study assessed the effect of smoking on the expression of TERT in the bladder. The present study's findings showed that exposure to cigarettes and e-cigarettes increased the expression of TERT in rat bladder. Previously, some studies have assessed the interaction between TERT and smoking in other cancers. Capkova et al., assessed the rate of TERT expression in the bronchial mucosa of heavy smokers. The findings showed that the expression of TERT in bronchial biopsies with moderate or severe dysplasia and carcinoma was significantly higher than the normal and mild dysplasia. However, a significant association was not found between smoking status and TERT expression (21). In Lotfi et al.,'s study, there was an inverse association between smoking status and TERT expression in the skin dermis (22). Therefore, there is a lack of data on the interaction between TERT expression and smoking, and more studies are needed to conclude.

FGFR3, a member of the transmembrane receptor kinase for the FGF family of ligands, plays a vital role in cancer cell proliferation, epithelial-to-mesenchymal transition (EMT), migration, and invasion (23). The findings of studies about the impact of cigarettes on the expression of FGFR3 are contradictory. In the present study, the expression of FGFR3 increased in rat bladder in both cigarette and e-cigarette groups. Du et al., assessed the effect of nicotine as the leading tobacco component on the expression of some miRNAs in non-small-cell lung cancer cell lines, and the findings showed that nicotine significantly increased the mRNA and protein expression FGFR3  (24). Another study reported that the FGFR3 expression level was significantly higher in urothelial carcinoma tissues than in normal tissues, and overexpression was associated with cigarette smoking (25). In contrast with previous studies, Tomlinson et al. reported no significant association between FGFR3 expression and smoking habit in bladder cancer patients (26). So, more studies need to explore the association between FGFR3 expression and cigarettes. 

VEGF, an endothelial cell-specific mitogen, is a necessary factor for inducing angiogenesis. We found the upregulating of VEGF expression after exposure to cigarettes and e-cigarettes. To assess the association between cigarettes and patterns of VEGF expression, Rahmani has compared the patients with transitional cell carcinoma and control with inflammatory lesions of the bladder. They reported no significant association between smoking status and VEGF expression. these results might support the hypothesis that certain carcinogens derived from cigarette smoking may induce VEGF mutations and apoptosis, which are involved in the early steps of bladder carcinogenesis  (27).

PTEN is one of the most frequently mutated genes in human cancer. PTEN tumor-suppressive activity regulates cell growth, proliferation, and genomic stability. The PTEN expression decreased in cigarette and e-cigarette groups in the current study. Brait et al. investigated molecular events that lead to urothelial cell carcinoma, and for this purpose, they exposed the human bladder epithelial cells to 0.1% cigarette extract for four months and found a slight decrease in the expression of PTEN  (28) that our results were consistent with this study.

The tumor-suppressor protein p53 is a crucial regulator of cell apoptosis, classically associated with the genesis of invasive urothelial carcinoma. We found a decrease in P53 expression after exposure to cigarettes and e-cigarettes; except for female rats in the e-cigarette group, the expression of P53 increases after exposure. This result showed that e-cigarette exposure induced apoptosis in the female rats in the study period, and more studies need to assess the reason for the different results between the two groups. Cheng-Hsun Wu et al. showed that cigarette smoke increased the cellular levels of phospho-p53 in rat terminal bronchioles  (29). However, another study reported that the P53 expression was unchanged after exposure to cigarette smoke extract (28). The strength of the current study is that it is the first study to compare the effect of cigarettes and e-cigarettes on the expression of TERT, FGFR3, PTEN, P53, and VEGF in rat bladder.

Conclusion

We found that both cigarettes and e-cigarettes changed the expression of genes involved in the development of bladder cancer, but no significant differences were found between cigarettes and e-cigarettes. Therefore, the e-cigarette is not a good alternative to cigarettes and it was suggested that quitting smoking is better than replacing cigarettes with electronic cigarettes.

Author's contributions

All authors contributed equally.

Acknowledgments

Special thanks to the urology Research Center, Tehran University of Medical Sciences, Tehran, Iran.

Ethical considerations

All animal experimentations and the study design were approved by the Ethical Committee of the Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1399.940).

Competing interests

All authors claim that there is no conflict of interest.

Funding

There is no funding.

Data Availability

Information, data, and photos will be provided if requested.