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ORIGINAL ARTICLE
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Overexpression of Beclin1 gene leads to reduction of telomerase activity in MDCK cells and enhances apoptosis


1 Department of Animal Biology, Faculty of Biological Science, Kharazmi University, Tehran, Iran
2 Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
3 WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran, Tehran, Iran

Correspondence Address:
Homa Mohseni Kouchesfahani,
Department of Animal Biology, Faculty of Biological Science, Kharazmi University, Tehran
Iran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_265_17

 > Abstract 


Background: Telomeres through maintaining chromosomal integrity have key roles in the cell life span. The autophagy is typically a pro-survival process and important for maintaining cellular homeostasis. Conversely, in some conditions, autophagy acts as caspase-independent cell death program. Beclin1 gene plays a principal role in the initiation of autophagy.
Objective: The aim of this study was to evaluate the effect of autophagy induction via recombinant Beclin1 on telomerase activity and programmed cell death (apoptosis) in MCDK cells.
Materials and Methods: The recombinant Beclin1-pcDNA3.1(-) was transfected into MDCK cells. Next, the autophagy information was detected by LC3II staining as autophagy marker using flow cytometry. The telomerase activity was measured by telomeric repeat amplification protocol method in MDCK cells. To detection of the cell death in MDCK cells, apoptosis assay was done through Annexin V staining method.
Results: The results of flow cytometry analysis indicated that following overexpression of Beclin1 gene, the percentage of the LC3II was 16.08% compared with control group (0.48%). Following induction of autophagy, telomerase activity reduced 10 folds in comparison with the control group. The rate of apoptosis in transfected MDCK cells increased up to 12.74%.
Conclusion: Crosstalk between telomerase, autophagy, and apoptosis may determine the fate of the cancer cell aging. Hence, manipulation of autophagy may create a novel area to design new compounds and combination therapy to shorten the cancer cell survival.

Keywords: Apoptosis, autophagy, Beclin1, MDCK cells, telomerase activity



How to cite this URL:
Taji F, Abdoli A, Baesi K, Sheikholeslami F, Kouchesfahani HM. Overexpression of Beclin1 gene leads to reduction of telomerase activity in MDCK cells and enhances apoptosis. J Can Res Ther [Epub ahead of print] [cited 2019 Nov 21]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=268948




 > Introduction Top


Telomeres are repeated units of guanine rich at chromosome ends.[1] Telomeres are key regulators of both cell life span and chromosomal integrity.[2] Furthermore, telomere protects chromosomes from damage and degradation. Telomerase, a reverse transcriptase enzyme which added TTAGGG tandem repeats onto the end of eukaryotic chromosomes, is strongly associated with cancer. Observations showed that telomeres and telomerase have important roles in suppressing malignant transformation.[2] Telomerase activation has been seen in 98% immortal cells and in 90% of malignant tumors.[3],[4] Telomerase has key roles in carcinogenesis.[5]

The accumulation of damaged cellular components and misfolded proteins has deleterious effects on cellular homeostasis and on tissue integrity. The defective components and molecules can disrupt cellular homeostasis directly or by interfering with the function of organelles, which leads to further damage and dysfunction. This progressive reduction in cellular integrity resulting in aging, disease, and ultimately, to cell death.[6] Autophagy plays a key role in tumor suppression in early stages and cell aging by engulfing the malfunction proteins and organelles. Cancer genesis is genetically connected to autophagy defect suggesting that autophagy is a true tumor suppressor pathway.[7],[8] To induce autophagy, Beclin1 contributes in the initial steps of autophagosome formation.[9] The Beclin1 autophagy initiator gene regulates negatively tumor genesis.[10]Beclin1 is monoallelically deleted in up to 70% of types of human cancers.[11]

Due to disability to adapt under stressful conditions, autophagy leads to apoptosis or necrosis pathway.[12],[13] It has been shown that autophagy contributes in interaction with apoptosis process via completing the apoptotic caspases, so if principal caspases are incapacitated, autophagy can progress the process of apoptosis cell death. Therefore, depending on the conditions, autophagy can have either preventive effects or supportive in cell survival.[14] Molecular regulators are involved in crosstalk between cell death types. One of the central molecules that play important role in autophagic and apoptotic machinery is Beclin1.[12],[15] Beclin1 is composed of a BH3 domain and is in interacting with Bcl-2 family proteins, thereby inhibiting the formation of Beclin1-PI3K3 complex and autophagy induction.[16] Protein such a BAD affects Bcl-2/Bcl-xL and Beclin1 complex and breaks it. Therefore, disruption of Bcl-2-Beclin1 complex stimulates autophagy.[17] In overexpression of Beclin1 gene,[18] autophagy leads to suppressing tumor cell life span, possibly through activating apoptosis[19] or possibly through the inability of cells to contribute in nonspecific bulk degradation of the contents of the cell.

Apoptotic cell death represents an evolutionary pathway of cell suicide. Deregulation of apoptosis leads to diseases such as cancer in which apoptosis is inhibited. Moreover, cancer genesis is characterized by the telomerase inhibition. A recent publication showed a close relationship between telomerase enzyme activity and apoptotic cell death. First, Bcl-2 gene is the common modulator of both telomerase activity and apoptosis pathway. In human cancer cells which have low expression of Bcl-2 gene, telomerase activity is increased via bcl-2 overexpression.[20] Second, in the study of Fu et al. on anti-apoptotic role of telomerase, showed that telomerase has key roles in suppressing apoptotic signaling pathways.[21] Third, direct inhibition of telomerase activity significantly enhances telomere shortening and apoptosis.[22],[23] However, little is known about how the autophagy effects on telomerase activity and cell death. Therefore, the present study evaluated the rate of telomerase activity in association with autophagy induction and the rate of apoptosis in MDCK cells.


 > Materials and Methods Top


In this study, gene and primers were synthesized by Eurofins MWG, Germany. MDCK cells were obtained from Pasteur Institute of Iran. Rabbit polyclonal anti-LC3B antibody (ab48394), goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgG (ab98466) antibody, and Beclin1 gene (ab55878) were supplied by Abcam. BamHI and Xba1 restriction enzymes were obtained from Fermentas (MA, USA). Lipofectamine 3000, DMEM, fetal bovine serum (FBS), PUC57-kan, and pcDNA3.1 plasmids were from Invitrogen (CA, USA). Plasmid DNA extraction kit was supplied by Yekta Tajhiz Azma Company (Tehran, Iran). Annexin V FLUOS staining kit (11858777001) was purchased from F. Hoffmann-La Roche.

Cell culture and cell transfection

MDCK cells (1 × 105 cell/well) were grown in 6-well cell culture plates (Pasteur Institute, Iran) to 80% confluence. Cells were cultured in DMEM supplemented with 10% FBS, streptomycin (100 μg/ml), and penicillin (100 units/ml) in an incubator with 5% CO2. Cell viability was evaluated by trypan blue staining. Cells were transiently transfected with either Beclin1-pcDNA3.1(-) or empty pcDNA3.1 (-) using Lipofectamine 3000 as the transfection reagent according to the manufacturer's instructions. Transfected cells were incubated for 72 h in an incubator.[24]

Detection of autophagy induction using the autophagy marker protein LC3

To fix the MDCK cells, these cells were placed in formaldehyde with the concentration of 0.01% (v/v), for 10–15 min in 48 h posttransfection. Then, using Tween 20 (0.5% v/v in phosphate-buffered saline [PBS]), cell membranes were permeabilized. Rabbit polyclonal anti-LC3B antibody was added. Next, cells were shaken for 1 h at room temperature. Finally, cells were washed with PBS and incubated with secondary antibody (FITC) for 1 h. By evaluating the percentage of stained LC3-II-positive cells, the formation of these structures was measured. Flow cytometry was used for delineating of resulting graphs.[25]

Telomeric repeat amplification protocol assay

MDCK cells (106) were transfected with Beclin1-pcDNA3.1 (-) or PCDNA3.1 (-) vectors. MDCK cells were harvested 24 posttransfection. To do quantitative polymerase chain reaction (PCR), cells harvested 48 h posttransfection.[26] Then, cells were lysed in 200 μl of CHAPS lysis buffer (0.5% CHAPS, 5 mM mercaptoethanol, 10 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM MgCl2, 1 mM EGTA, and 0.1 mM PMSF) and incubated on ice box for 30 min. After the preparation of the cell lysates, lysates were centrifuged at 12,000 g for 20 min. Then, the resulting supernatants were collected. Test samples received both oligonucleotide TS and ACX or only one of these primers (TS and ACX are substrate and reverse primer, respectively).[27] To conduct real-time (RT) TPAP, in all samples, cell lysates were exposed to SYBR Green RT-PCR. The reaction mixture was prepared in a final volume of 25 μl of SYBR Green solution (PE Applied BioSystems), 12.5 μl of PCR buffer, 5 μl DEPC water, 1 U/μl of T4 DNA ligase, and 1 μl of oligonucleotide TS (0.1 μM). To activate telomerase enzyme in lysate samples, the reaction mixture was placed at 25°C for half an hour. Then, 1 μl of ACX primer and 1 U/μl of Taq polymerase enzyme were added to experimental groups, respectively. To activate Taq polymerase, RT-PCR was done at 95°C for 5 min using a Rotor-Gene 6000 Series System. RT-PCR experiment was continued by 40-cycle reaction (95°C for 20 s, 50°C for 30 s, and 72°C for 90 s).[28] Then, the produced fluorescence signals were analyzed, and telomerase activity in experimental groups was evaluated according to threshold cycle (Ct).[28] All samples were analyzed at least in duplicate.

Evaluating of apoptosis by flow cytometry

After 72 h posttransfection, the adherent cells (1 × 105 cell/well) in 6-well culture plates were trypsin zed and washed twice with PBS and then centrifuged (670 ×g, 5 min, RT). Then, resulting plates were re-suspended in PBS (400 μl). The transfected cells were received 100 μl of incubation buffer with 2 μl of annexin in the concentration of (1 mg/ml) and 2 μl of propidium iodide (PI) (1 mg/ml). Control groups only were received 100 μl of incubation buffer. After the incubation of cells in dark for 30 min, apoptosis rates were analyzed using flow cytometry without washing.


 > Results Top


Transfection of Beclin1 induces autophagy in the MCDK cells

Microtubule-associated proteins 1A/1B light chain 3B (LC3) is the most widely used marker of autophagosome formation. After LC3II staining with FITC-conjugated antibody, the LC3-positive cells were quantified via flow cytometry [Figure 1]a and [Figure 1]b. Beclin1 induced the accumulation of the LC3-positive dots in transfected cells in comparison with control group. In total, more than 16% of the cells were LC3 positive in transfected cells [Figure 1]a. In nontransfected cells, the LC3-II expression level was (0.48%) as shown in [Figure 1]b.
Figure 1: After 72 h transfection, overexpression of Beclin1 gene induced the accumulation of the LC3-positive dots in transfected cells in comparison with control group. In total, more than 16% of the transfected cells were LC3-II positive (a). In nontransfected cells, the LC3-II expression level was <1% and equal to 0.48% (b)

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Telomerase activity is inhibited in MDCK cells

Telomeric repeat amplification protocol (TRAP) assay is a number of several methods for determining telomerase activity. MDCK cells were harvested and lysed in CHAPS lysis buffer. Then, telomerase activity was evaluated using the TRAP assay. Accordingly, the values of the cycle threshold were plotted versus the number of cycles. The results showed that telomerase activity was lower in the transfected cells. The values of the Ct were 24.69 and 27.78 in nontransfected and transfected cells, respectively [Figure 2]. Following the induction of autophagy, telomerase activity decreased for about 10 folds in comparison with the control group [Figure 2].
Figure 2: SYBR Green real time-telomeric repeat amplification protocol assay was used for evaluating of telomerase activity in MDCK cell lysates. The fluorescence signals of SYBR Green were shown on the y-axis, against the cycle number on the x-axis. The values of the threshold cycle were 24.69 and 27.78 in nontransfected and transfected cells, respectively. The fluorescence amount detected for the first time at the top of the baseline is considered as the cycle threshold (ct)

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Overexpression of Beclin1 increases apoptosis in the MDCK cells

Apoptosis was checked through PI staining and flow cytometry. MDCK cells were transfected with empty plasmid considered as the blank control. The rate of apoptosis was more in transfected cells than in control [Figure 3]a and [Figure 3]b. The apoptosis analyses were assayed in independent experiments. The rate of apoptosis in transfected MDCK was equal to 12.74% and in control was equal to 0.07%.
Figure 3: Detection of apoptosis by flow cytometry. The induction of Beclin1 increased apoptosis in MDCK cells. The rate of apoptosis in transfected and control MDCK cells was 12.74% and 0.07%, respectively (a and b)

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 > Discussion Top


Evidence point to the role of autophagy as a key factor in the development of carcinogenesis and the response of tumor cells to life span longevity. The maintenance of quality control of the cell is one of the autophagy roles which is necessary for enhancing cell survival.[29] Autophagy maintains organisms against aging and diseases, autophagy maintains organisms against aging and diseases.[8] However, the function of autophagy in carcinogenesis is controversial. Autophagy inhibitors and promoters inhibit tumor genesis by mechanisms that are not clear.[30],[31] The results of studies show that there is a paradox in the function of autophagy. The study of Liang et al. showed thatthe existence of Beclin1 leads to decreased apoptosis in the central nervous system.[10] The study of Wang showed that the depletion of Beclin1 gene led to the activation of apoptosis.[32] These results are in contrast to the results of our study. Our results demonstrate that following the overexpression of Beclin1 and the reduction of telomerase activity, apoptosis pathway was activated.The expression of exogenous Beclin1 gene has prevented proliferation and tumor genesis in MCF-7 cells.[10]

Beclin1 led to the incidence of types of cancers including liver cancer, lung cancer, and lymphoma in transgenic mice.[33],[34] Possible function of autophagy in the pathogenesis of cancer has been shown by reducing the expression of the Beclin1 gene in lung cancer.[35]

Little is known about the function of autophagy in the tumor genesis of MCDK cells. With considering the possibility of the effect of the Beclin1 gene on the production of MCDK cell apoptosis, we assumed that Beclin1 upregulation may help MDCK cells to affect the organ's normal clearance mechanism by apoptosis induction. This would contribute to the promotion of cell death. Therefore, it appears that Beclin1 may have a different function in the regulation of apoptosis pathway and need to be evaluated with more details.

The direct role of Beclin1 in triggering apoptotic signaling has been suggested with binding this gene to Bcl-2 family members.[36],[37] In addition, this appears that several molecules are involved in triggering of apoptotic signals. Protein such as BAD affects Bcl-2/Bcl-xL and Beclin1 complex and leads to inhibit the interaction of between Beclin1 and Bcl-2 resulting in break it.[38] C-terminal segment of Beclin1 gene moves to mitochondria, and cytochrome c is release and mediates pro-apoptotic effects of Beclin1 gene. Calpains affect N-terminal Atg5 cleavage. Then, it translocates from the cytosol to mitochondria, and apoptotic cell death is triggered. The Atg3 molecule has a pro-autophagic activity and is principal target of caspase-8. Atg3 cleavage inhibits its pro-autophagic activity. In conditions that death receptor apoptosis is induced, autophagy is inactivated.[39] Atg4D is a direct target of caspase-3 cleavage which can induce the delipidation of the LC3 molecule.[40] Autophagic signaling promotes an interaction between some of ATG(s) and the activation of caspase-8. Then, caspase-8 can affect cleavage the regulator of apoptosis and necroptosis (RIPK1, a serine/threonine kinase).[41],[42] RIPK1 contributes in a negative feedback loop which is limiting for autophagic pathway.[43]

Age-related telomere length reduction threatens chromosome integrity of aging tissues.[44] Progressive decline of telomere lengths with age has been shown in human tissues and mice.[45],[46] Studies support the notion that the loss of function telomerase affects age-related telomere length reduction. Telomerase mutations lead to shorter telomeres and premature aging phenotype.[47]

It appears that in our study, apoptosis activation may be resulting from the loss of function telomerase activity in cells. A study of Liu et al. demonstrated that downregulation of telomerase activity and apoptosis pathway is time and concentration-dependent pathway.[48] Telomerase activity regulates at multiple levels, and its downregulation is possibly in the upstream event of apoptosis signaling. However, the distinct relation of telomerase activity and apoptosis is not clear. Another possibility is that both telomerase downregulation and apoptosis induction are an independent event.[48]

Accumulating evidence suggests that telomere maintenance is an important factor in life span longevity and genome instability.[45],[49] Genome stability, proliferation, and survival in cells are regulated by pathways such as DNA repair and programmed cell death.[30] Previous studies have established the relationship between genome instability and cell death control in carcinogenesis.[50]

Apoptosis in cells which have dysfunction telomeres is resulting in the existence of telomere dysfunction or the existence of chromosomal breaks induced in cells that continue to divide with a fused chromosome. Therefore, in the lack of chromosomal fusion, dysfunctional telomeres possibly are considered as DNA double-strand breaks and display an apoptotic response.[51] Double-strand breaks will not be repaired in the lack of nonhomologous end-joining proteins resulted in an induction of apoptosis response.

The use of telomerase activity assays in tumor life span remains an area of further attention. Therefore, targeting telomeres can be considered as a new approach in life span longevity and the development of mechanisms for cancer therapeutics.


 > Conclusion Top


Key regulators seem to determine the thresholds of cell death process in cellular reaction and may have controversial roles in physiological function versus pathological functions. Telomerase is essential for the maintenance of tumor cell life span. Therefore, precise quantification of this enzyme is a useful target for the design of antitelomerase agents in the future.

Acknowledgments

The authors thank Mrs Moghadam (cell Bank) for the gift of MDCK cells.

Financial support and sponsorship

This work was financially supported by a grant from Kharazmi University (Tehran, Iran). We would like to thank Pasteur Institute of Iran, Tehran, for his collaboration and support during this research.

Conflicts of interest

There are no conflicts of interest.



 
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