Genotyping determination of GSTM1 in breast cancer patients
The distribution of GSTM1 genotype in the breast cancer tissues and ANCT tissues were determined by standard PCR as shown in figure 1A and table 1. The frequency of null GSTM1 genotype was significantly higher (0.733) in breast cancer tissues samples compared with the ANCT samples (0.134) (p=0.004), Further, we categorized the breast cancers patients based on their age, it was revealed that the null-GSTM1 genotype frequency was significantly higher in patients with age less than 50 (<50) years as compared with patients of greater than 50 (>50) years age of patients (0.705 vs 0.582, p=0.007). These data indicate that mutation in GSTM1 genotype has significant association with early development of breast cancer in Pakistani population. In addition, HPLC result determined that the level of endogenous glutathione (GSHt, GSH, GSSG) were decreased in patients with age less than 50 (<50) years compared with patients having age greater than 50 (>50) years as shown in figure 2.
Determination of GSTM1 expression in breast cancer patients
Western blotting and RT-PCR were performed to evaluate the protein and mRNA expression levels of GSTM1 in both tumor and control individual tissues. The relative expression of GSTM1 mRNA transcript was found to be significantly decreased (p=0.001) in the tumor tissues samples compared to the ANCT samples, irrespective of the histological and disease status of the patient (Figure 1B). Furthermore, the concentration of GSTM1 proteins were determined by western blot. The result concluded that the protein concentration of GSTM1 was significantly decreased in breast cancer tissue samples (p=0.003) as compared with ANCT samples as shown in figure 1C. These result revealed that downregulation of GSTM1 genotype promotes EMT pathway that enhanced the expression of tumor metastasis and invasion biomarkers which is mainly associated with the development and progression of breast carcinomas.
Determination of GSH concentration among patients with different age groups
Glutathione (GSH) plays crucial role in a multitude of cellular processes, including cell proliferation, differentiation, and apoptosis and alteration in GSH homeostasis are participating in the development and progression of different diseases, including cancer. The deficiency of GSH or decrease in the ratio of GSH/glutathione disulphide (GSSG) leads to enhanced susceptibility to oxidative stress implied in the development of cancer. Therefore, the endogenous GSH (GSHt, GSH, GSSG) concentrations were determined by HPLC and compared between two study cohorts of breast cancer, <45 and >45 years. Interestingly, in both study cohorts, the GSHt concentration was higher in the control tissues (135.206±39.031 and 138.59±38.438 respectively) compared to that of tumor tissues (22.478±4.792 and 30.468±5.758 respectively). Statistical analysis showed that GSHt concentrations were significantly associated with both tissue types (p=0.003, p=0.005), while GSH and GSSG concentrations were also decreased in tumor tissues of both study cohorts, but no significant difference was observed (p>0.05) between tumor tissue samples and ANCT samples (Figure 3, S Table 1). These data indicated that endogenous GSH, especially GSHt was significantly downregulated in tumor tissues in both study cohorts.
GSH concentration with premenopausal and postmenopausal status of breast cancer patients
Further, the endogenous concentration of GSH were investigated among premenopausal and postmenopausal breast cancer patients. Of the total study cohorts (n=198), 115 belonged to the premenopausal group and 83 belonged to the postmenopausal group. The GSHt concentration was significantly lower in the tumor tissues samples of premenopausal group (25.52±4.361) compared to ANCT samples (133.479±32.372, p=0.006). Similarly, significant reduction in GSHt was observed in the tumor tissues samples of postmenopausal group (30.602±7.422) compared with the ANCT samples (145.183±51.330, p=0.009). In addition, the concentration of GSH and GSSG were also significantly decreased in premenopausal tumor tissues samples (p<0.05), while no difference has been observed in postmenopausal tumor tissues samples (p>0.05) as compared with the ANCT samples. The data obtained suggests that GSHt was significantly downregulated in the tumor tissues samples of both premenopausal and postmenopausal patients (Figure 4, S Table 2).
Determination of GSH among different stages and grades in breast cancer patients
Moreover, the endogenous GSH levels were determined in different stages and grades of breast cancer. According to stages wise stratified, 86 samples belong to stage I, 64 belong to stage II and 48 belong to stage III of the study subjects as shown in table. The data reveal that the GSHt levels were decreased in breast cancer tissue samples in all tumor stages (I, II and III) (24.499±24.527, 29.838±7.459, 28.556±14.428) compared to ANCT samples (134.955±34.488, 110.811±41.883, 136.776±96.714). The results obtained were statistically more significant in stages I and II of breast cancer (p=0.014, p=0.003) as that of stage III in breast cancer patients (p=0.031). The concentration of GSH, and GSSG was decreased as advancement in stages of breast cancer, however, the results were statistically not significant (p>0.05) (Figure 5A, S Table 3). Further, their levels were also correlated with different grades of breast cancers. Of total study cohorts (n=198), 78 grade I, 63 grade II, and 57 individuals belong to grade III. Similarly, the levels of GSHt were higher in all grades of tumor tissues samples (211.675±122.27, 128.452±29.468, 142.11±82.047) compared to ANCT samples (19.013±7.762, 26.19±4.194, 26.798±11.984). These results were also more significant in grades I and II of breast cancer patients (p=0.03, p=0.036) as compared to grade III individuals (p=0.05). Hence, the concentration of GSH and GSSG has no significant correlation with tumor grades (Figure 5B, S Table 4).
Downregulation of GSTM1 promotes tumor proliferation, metastasis and invasion in breast cancer patients
Present study further evaluate the expression of various biomarkers, including tumor, proliferation, and invasion and metastasis in the cancer tissue samples and ANCT samples of breast cancer patients. The mRNA expression showed that tumor biomarkers, including HER2 and CEA were significantly upregulated in tumor tissues (p=0.001, p=0.03) as compared with ANCT. Interestingly, upregulation of HER2 expression is more significant than CEA (Figure 6A). Similarly, tumor proliferation biomarkers were also analyzed in breast cancer tissues using RT- PCR. The mRNA expression revealed that proliferation markers, including Ki-67, P-AKT, and Bcl2 were significantly increased in tumor tissues samples compared to ANCT samples (p=0.006, p=0.009, p=0.014) (Figure 6B). The result was highly significant and show that the tumor tissues has high rate of proliferation. For further validation, tumor metastasis and invasion were determined in the tumor tissue samples and ANCT samples. The results revealed that the expression of metastasis and invasion biomarkers, including MMP-2 and MMP-9 were significantly increased in breast cancer tissue samples verses ANCT samples (p=0.007, p=0.009) (Figure 6C).
Downregulation of GSTM1 induces Epithelial to mesenchymal cell transition pathway
Epithelial to mesenchymal cell transition (EMT) plays a crucial role in appropriate morphogenesis during development. Disregulation of this process has been concerned as a key events in fibrosis and the progression of carcinomas to a metastatic state. However we analyzed EMT process in breast cancer tissue samples and ANCT samples. Both RNA and proteins were isolated to determined EMT pathway through RT-pcr and western blot. We finalized that downregulation of GSTM1 induced the mRNA expression of mesenchymal marker i.e vimentin (*p=0.019) Snail (**p=0.0074) and Fibronectin (**p=0.0089) via decreased the expression of epithelial marker i.e E-cadherin (**p=0.009,), MUC-1 (**p=0.0082) and lamine (**p=0.0097) in breast cancer tissue samples as compared to their ANCT samples. In addition, Present study also determined that the protein expression of mesenchymal markers vimentin, snail were increased (**p=0.006, **p=0.0027) while the expression of epithelial marker i.e E-cadherin, MUC-1 were decreased (**p=0.0057, **p=0.0078) in breast cancer tissue samples as compared to ANCT samples using GAPDH as negative control as shown in figure 7. Each results were repeated as triplicate.