In the present study, H.pc-TSGA10 cells showed a higher expression of autophagy-related genes compared with H.pc cells under normoxic and hypoxic conditions. Acridine orange staining demonstrated higher acidic vesicles due to higher rate of autophagy in H.pc-TSGA10 cells in compared with H.pc cells under various conditions. Increased expression of BAX and BAX/Bcl2 ratio as an apoptosis index, increased cleaved caspase-3 level, and decreased expression of BCL-2 confirmed higher rate of apoptosis in H.pc-TSGA10 cells compared with H.pc cells in both normoxic and hypoxic conditions. Moreover, cell viability was significantly decreased in both stably and transiently transfected cells with pcDNA3.1-TSGA10 in the presence or absence of autophagy and apoptosis inducers under normoxic and hypoxic conditions.
Autophagy contributes in determining overall fate of the cells (23). Autophagy can used as a practical target in the clinic to destroy cancer cells by direct induction of type II programmed cell death (11). Defective function of Beclin 1, as a known regulator of autophagy and a tumor suppressor gene has been observed in several cancers. Increased expression of Beclin1 can induce autophagy and consequently prevent tumor development. The BCL-2/Adenovirus E1B 19kDa-intracting Protein 3-Like (BNIP3) and BNIP3 like (BNIP3L/NIX) genes, belonging to BCL-2 (BH3-Only) family, can induce autophagy-mediated cell death. BNIP3 and BNIP3L competes with Beclin1 in binding to BCL-2 or Bcl-xL and hence can lead to autophagy and mitophagy by separating Beclin1 from BCL-2 (24). Previous investigations have also illustrated that apoptosis inhibitors can induce autophagy by increasing level of Beclin1 and inhibiting formation of Beclin1/BCL-2 complex (25, 26). BNIP3L gene product plays important roles in the pathobiology of various diseases such as cancer. The modulated expression of BNIP3L during tumor hypoxia can directly influences tumor growth. Expression of this protein is required for an optimum autophagy induction in hypoxic conditions needed its BH3 domains assists in competitively separating Beclin1 from Beclin/BCL-2 and Beclin/BCL-xL complexes (27–29).
Roghanian A, et al. have reported an inhibitory interaction between TSGA10 and vimentin (30). In addition, Wang RC, et al. have indicated that a drastic decrease in vimentin is associated with higher Beclin 1 levels which can consequently lead to increased autophagy (31). HIF-1α regulates autophagy by the induction of the expression of certain pro- and anti-apoptotic proteins such as BNIP3, BNIP3L and BCL-2 (32).
The ATG5 is an inducer of autophagy-dependent cell death, which can lead to LC3I to LC3II conversion. The LC3II contribute in autophagosome formation, thus, the expression level of this protein is associated with rate of autophagosome formation in mammalian cells (33). Our findings confirmed a significant increased expression of autophagy-related genes such as ATG5 and LC3II in H.pc-TSGA10 cells compared with H.pc cells, which indicated that overexpression of TSGA10, could significantly induce autophagy. ATG5, a simulator of autophagy-dependent cell death, plays a significant role in apoptosis as a downstream effector of caspases. Increased ATG5 activates apoptosis through calpain activation (33, 34).
In a previous study, we demonstrated that TSGA-10 overexpression caused decreased expression of angiogenic factors such as VEGF, CXCL12 and CXCR4 (3). Domigan, et al. showed that decreased levels of VEGF promoted mitochondrial fragmentation and lower glucose metabolism finally resulting in autophagy induction and cell death. Their findings showed that nhibiting VEGF expression led to increased autophagy by mean of rising FOXO activity (35). In addition, another study showed that increased bFGF levels decreased autophagy by inhibiting LC3 II (36). Hashmoto et al. reported that autophagy could be restricted by increased levels of progesterone which upregulates the expression of CXCL12/CXCR4 (37). Based on these findings, it seems that inhibiting angiogenesis via TSGA10 overexpression induced autophagy in H.pc cells (Fig. 8)
According to a previous study, upregulation of TSGA10 induced autophagy through induction of P21. Induced P21 consequently inhibits BCL-2 (38). Therefore, TSGA10 overexpression can indirectly promote autophagy and apoptosis by BCL-2 inhibition. Tumor cells overexpressing BCL-2 are quite resistant to apoptosis through downregulation of pro-apoptotic proteins, Bak and BAX. Considering important roles of BCL-2 in regulating apoptosis and autophagy, its inhibition would be promising in order to sensitize tumor cells to programmed cell death (PCD) (39, 40).
Furthermore, TSGA10 overexpression can induce cell cycle arrest at G1/S through increasing levels of Rb, P53, P21, p16 which consequently leads to apoptosis (41). In our study, decreased cell viability following TSGA10 overexpression was a consequence of activated apoptosis, which was mostly due to cell cycle arrest at G1/S and was partly due to BCL-2 inhibition. Based on a previous study, TSGA10 overexpression decreased CXCL12, MMP2 and MMP9 expression by inhibiting HIF-1α (3). Decreased expression of MMP2 and MMP9 leads to CytC release and changes the expression levels of BAX and BCL-2 which consequently provokes apoptosis (42). In addition, decreased expression of CXCL12 and CCL25 has shown lead to higher rate of apoptosis (43, 44). All the above-mentioned findings affirm that TSGA10 overexpression can induce apoptosis via regulating multiple apoptotic targets (Fig. 8).
The TSGA10 gene, located on chromosome 2 (q11.2), consists of 19 exons and is associated with the sperm tail. Human TSGA10 is constituted of 698 amino acids. In mice, this protein is expressed in normal cells and tissues such as cerebellum, bone marrow, thyroid, brain, eye bulb, cecum, hematopoietic stem cells and testis. TSGA10 overexpression has been detected in various conditions such as liver carcinoma, ovarian tumors, prostate, bladder, and colon cancer, malignant melanoma, cutaneous T-cell lymphoma and as well as autoimmune diseases. (1, 13, 45).
It has been widely accepted that TSGA10 plays a significant role in the spermatogenesis. In testis, falling oxygen level to less than 1% (hypoxic stress) compared with 2–9% in normoxic condition for many tissues of mammalian species is a disturbing agent for fetus development and is a risk factor for many pathologic conditions such as solid tumor, coronary artery ischemia and brain damages. Regarding high rates of cell proliferation and differentiation during spermatogenesis, being hypoxic causes dangerous condition and increased risk of tumor formation in testis tissue (15, 30, 46–48). Thus, expression level of HIF-1α and its downstream effectors regulating various cellular mechanisms is higher in testis in compared to other tissues. HIF-1α overexpression has been shown in various cancers. Cancer cells adapted to hypoxia are usually resistant to apoptosis and so, cancer treatment requires more aggressive strategies.
In testis cells with hypoxic microenvironment, it is crucial to identify the mechanisms regulating cell proliferation, cell cycle and survival in order to balance cell numbers and consequently, prevent testis cancer. TSGA10 can regulate this balance by inhibiting HIF-1α, which is expressed in higher levels in hypoxia compared with normoxia. Target genes of HIF-1α contribute in various cellular mechanisms such as apoptosis, angiogenesis, autophagy, glucose metabolism, erythropoiesis, tumor development and so on. In tumor development, HIF-1α plays an important role in establishing a balance between oxygen need and supply. HIF-1α is considered as a substantial molecule for adaptation of cells to stress like hypoxia by regulating expression of hundreds of genes. Inhibition of this molecule would hinder excessive cell proliferation and growth and thus could prevent cancer development. (3, 49, 50). Our previous investigation showed that a 55-kDa fragment of TSGA10 protein could inhibit HIF-1α:P300/CBP complex formation by binding to its TAD-C and PAS-B domain leading to HIF-1α inactivation. Therefore, this interaction indirectly resulted in BCL-2 inhibition (32).
Based on molecular docking results, eight out of the nine residues of BCL-2 which participate in its interaction with TSGA10 locate on its BH2 domain, which is shown to be required for the interaction with BAX and for anti-apoptotic activity (51). Thus, the possible molecular mechanism underlying the inhibition of BCL-2 by TSGA10 may be through covering its specific domain which mediates its oncogenic activity. Analysis of energies revealed a great negative free energy of TSGA10/BCL-2 interaction, where electrostatic attraction plays the major role in keeping the two proteins bound to each other. BCL-2 may attenuate inflammation by interaction with NLRP1, impairing NLRP1-inflammasome activation (52). This binding is via the putative loop between motifs BH4 and BH3, which is not involved in the interaction with TSGA10.
Although the accurate function of TSGA10 gene is still not clearly understood, it seems that in addition to spermatogenesis, it is also contributed in autophagy and apoptosis. The present study is in agreement with those recognizing TSGA10 as a tumor suppressor gene (3). Our previous study illustrated that TSGA10 overexpression could inhibit angiogenesis and tumor cell proliferation, migration and invasion by inhibiting HIF-1α (53). We demonstrated that in order to exerting antitumor effects of TSGA10 protein on cancer cells, its expression should reach a certain threshold. Thus, to apply TSGA10 as a tumor-suppressor, we used an expression vector with the highest rate of TSGA10 expression. In the present study, the role of TSGA10 in the induction of autophagy and apoptosis and decreasing cancer cell viability was shown. Since the TSGA10 is considerably expressed in various tumors, some studies have described it as a cancer testis antigen (CTA) (13, 15, 48). As a CTA, TSGA10 should be highly expressed only in testis, placenta and tumors, but it is widely expressed in normal human and mouse tissues. According to our present and previous studies, TSGA10 is not a CTA, but it is a tumor-associated marker with tumor suppressive function (3). This new function seems to be among cell regulatory mechanisms used to prevent tumor growth and development through autophagy and apoptosis inductions, thus, it might be considered as a promising candidate for cancer treatment and management.