AbstractBackground: Autophagy is a physiologic process in which double membrane vesicles engulf damaged proteins and organelles for delivering them to lysosomein order to degrade and recycle them via lysosomal digestion. Beclin1 is one of the basic proteins involved in initial step of autophagosome formation. In the current study, the effect of exogenous Beclin1 to induce autophagy and the effect of 3MA to inhibit of autophagy was assessed in Huh7.5 cells as an in vitro models of hepatocellular carcinoma.Material and method: The Recombinant pcDNA-Beclin1was transfected into Huh7.
5 cells. Also, the cell treated with 3MA. Next, the autophagy induction and inhibition was conducted via LC3 staining as a main autophagy marker using flow cytometry.Result: The result of this study suggest that the over expression of exogenous Beclin1 in Huh7.
5 cells elevated the autophagosome formation as shown by intracellular autophagosomal marker LC3-II staining for about 32.32 % and 3MA decreased it up to2% in compared with control cells in which the stained LC3-II was12.08.
Conclusion: Recombinant beclin1 may be used as a potential autophagy inducer agent and 3-methyl-Adenin inhibits autophagy formation in Huh7.5 cell. The staining autophagy formation marker LC3-II with specific antibody is a reliable method to measure autophagy activation via flow cytometry. Keywords: Autophagy, Flow cytometry, LC3-II Introduction:Autophagy is a preserved intracellular homeostatic process by which the cell cleans out different type of cytoplasmic debris(1). During the autophagy process, Beclin-1 and microtubule-associated protein 1 light chain3 (LC3) are connected with the initial steps of autophagosome formation and autophagosome maturation, respectively(2).
To start autophagy, Beclin 1 is an initiator complex that acts as a platform for nucleation step of autophagy. One of the key biological markers frequently used to show autophagy maturation in mammalian systems is the microtubule associated protein 1A/1Blight chain 3 (LC3). During elongation of the phagophore, cytosolic LC3-1 is conjugated to phosphatidylethanolamine to form LC3-II. LC3-II is recruited and inserted into the autophagosomal membrane(3). Finally, the delivery of autophagosomes to lysosomes and degradation takes place(4).Failure in autophagy cause to accumulation of intracellular macromolecules including unfolded and damaged proteins, damaged organelles, chromatin instability DNA .The most important mark to show the role of autophagy in tumor suppression is a study carried out on the interaction between Bcl2 and Beclin 1 proteins. Deletion of Beclin1 has been mounted in prostate cancer, breast, ovarian both in humans and mice.
Activation of PI3K-AKT-mTOR pathway that inhibits autophagy is a dominant condition for cancer culminating in tumor cell survival and proliferation In contrast, tumor-suppressor proteins such as RB, P53 and phosphates have a positive role on autophagy formation(5).Hepatocellular carcinoma (HCC) is an aggressive form of cancer and is the fifth most common cause of cancer death worldwide(6).The interaction between apoptosis and autophagy is remained as debated issue particularly in HCC. Hence, molecular mechanisms contributed to chemo resistance of HCC may result in improved clinical results.
Among the several parameters , therapy-induced autophagy indicate a novel approach of resistance to combat against cancer(7).Throughout dysplastic stagein hepatocytes, basal autophagy able to suppress tumor suppressor via eliminating newly damaged mitochondria and gnomically unstable cell to maintain genomic stability. Autophagy inhibitors lead to tumor suppression in tumor-forming stage in the HCC rat model, but result in tumor-promotion in dysplastic phase.
For this reason, the autophagy plays like two edge sward in the generation and progression of HCC based on the context of liver cells (8).Interestingly, evidence has been mounting that autophagy itself may be another cell death mechanism, termed programmed cell type II or autophagic cell death (ACD), emphasizing a potential pro-death role for autophagy ACD is characterized by the accumulated cytoplasmic vacuolization, absence of chromatin condensation, LC3 lipidation and caspase-independent apoptosis(9).Huh7.
5 is derivative of Huh-7 cells (is a human hepatocyte-derived carcinoma cell line) that shows significantly superior permissiveness to replication of HCV. Huh7.5 cells are the RIG-I pathway deficient cells, making them less sensitive to intracellular dsRNA throughout virus replication and eliciting an antiviral response(10).The manipulation of autophagy in Huh7.
5 can aid to clear the cross-talk between intracellular innate responses and viral immunopathogenesis. The aim of this study was to optimize the autophagy induction with recombinant beclin and autophagy inhibition using 3MA by staining go intracellular autophagosomal marker LC3-II using flow cytometry(11) . Material and MethodPlasmid constructionThe recombinant plasmid pcDNA3.1 containing Beclin 1was gifted by Dr. Asghar Abdoli (Pasteur Institute of Iran).The vector was transferred into Escherichia coli(E. coli) strain DH5a and the plasmid was purified from an overnight culture of E.
coli by using Miniprep Kit (Genetbio, Korea).(The Victor is 4,000 bp and the Beclin-1 gene is 1600 bp).Cell culture and treatmentOne day before transfection 2 × 105 Huh7.5 cells were seeded into in six-well culture plates in DMEM supplemented with 10% fetal calf serum 100 IU/ml penicillin and 100 mg/ml streptomycin and incubated in the presence of 5 % CO2 incubator.
After reaching 80% confluency, the growth medium was removed from cells and washed for two times with PBS buffer. In the next step, based on manufacturer protocol, the cells were transfected using Lipofectamine 2000(Sigma Company, Germany) mixed with 5µg of pcDNA-beclin 1. Also, to inhibit autophagy, Huh-7 cells were treated with5 mM of 3-methyladenine (3-MA)(12). Detection of autophagy induction by flow cytometryInduction of autophagy was verified via autophagosomal marker LC3-II detection using specific antibodies and flow cytometry.
Briefly, after fixation with 4% formaldehyde for 15 min and washed with PBS. Then the monolayer cells were permeabilized by adding 0.1% Triton X-100 in PBS for 15 minutes at 37° C?after removing Triton, the cells were covered by diluted primary antibody diluted in PBS containing 1% BSA for 1hr on ice. Finally, FITC-conjugated secondary antibody (Abcam, USA) was added and incubated at room temperature for 1h. The stained cells were studied by flow cytometry(Partec, Germany)(13). ResultsCell cultureHuh7.5 cells reached the desired confluency in terms of number and morphology after passages shown in Figure 1. Figure1: Inverted microscopic images of Huh7.
5 cells after 48 hours cultivation in DMEM with 10 % serum with a magnification of 40X Exogenous Beclin 1 able to induce autophagy in Huh7.5LC3 is the one of main reliable marker of autophagosomes formation. We tested the ability of Beclin 1 to trigger autophagy in Huh7.5 cells using monoclonal antibody through staining LC3-II. As can be seen in Figure 2, 32.32 % of the cells were LC3-II positive in the experiment sample in comparison with the LC3-II expression level in control cell was 12.08%, as shown in Figure 3. Figure2.
Autophagy induction by recombinant Beclin1.The staining intracellular marker of autophagy formation demonstrated that the 32.32 % of the cells were LCII positive when transfected with pCDNA-Beclin1 as autophagy inducer construct Figure 3Autophagy evaluation after empty pCDNA3.1 plasmid transfection as a control.
The Huh 7.5 cells were transfected with empty pCDNA3.1 stained with specific antibody to determine LC3II positive cells. Approximately, 12.08 % of cells were LC3II positive after staining Autophagy inhibition via 3MA3-Methyladenine (3-MA) is an autophagy inhibitor and selectively blocks PI3K. It reduced autophagy formation up to 2.2% as demonstrated in Figure 4.
Figure 4.Autophagy inhibition using 3-MA.The class III PI3K blocker 3-methyladenine (3-MA) is used to inhibit autophagic activation and it reduced autophagy formation up to 2.80 % DiscussionIn cancer cells, metabolic stress is because of inadequate nutrition or decreased storage Oxygen and increased require for quick cell division, induced autophagy because of cell search for energy source and metabolism alternatives. Also, autophagy may be a response to acquired cancer cells in cancer treatments which leads to drug resistance and cancer cell survival. Suppression of autophagy via drugs including 3MA make the cancer cells sensitive to a variety range of therapeutic models(14).
Parallel with this study, Kabeya Y and colleagues reported that conversion of LC3-I to LC3-II was repressed by 3-MA(15). These results verified the autophagy inhibitory effects of 3-MA on under normal conditions. In line with our studies, Tajo and et al. showed that Beclin 1 overexpression to induce autophagy in HeLa cells. They observed a number of LC3-positive cells by flow cytometry analyze is the HeLa cells transfected with pcDNA-Beclin 1expressed enhanced levels of LC3-II.
The Beclin 1 overexpression effect on autophagy formation in transfected cells was more than that of the control group. In total, 51.7% of the cells were LC3-II positive in the experiment samples(16). LC3 immunostained cells showed fewer numbers of punctuated stained cells on 3MA plus I2 treatment as compared to I2 treatment alone, suggesting disruption of autophagosome formation.
In addition, inhibition of autophagy with PI3K (3MA, LY294002) or H+/ATPase (bafylomycin) inhibitors enhance the cytotoxic response of I2. These data suggest that autophagy is acting as survival mechanism while extensive damage may be responsible for cytotoxic response in I2 treated cancer cells(17).Differential effects of the autophagy inhibitors 3-methyladenine and chloroquine on the control of autophagy on HL60 cancer cells, it was shown that as a result of treating cells with 3-methyl adenine, and then measuring using Western blot, there was no significant change in the LC3-ll bands, while in the cells incubated with chloroquine, a significant increase in the expression of this protein was observed. Based on these observations, it is hypothesized that depending on the stage of the autophagy process that is targeted for the drug, the type of response to the drug in the amount of expression of various genes involved in autophagy can be different(18).Demishtein A and et el, Reported when chloroquine or biflumycin A1 are cultured separately with neuronal coronal cells lead to autophagy inhibition by measuring and observing the LC3-ll protein(19). tri-methyl adenine(3-MA) can suppress proteolysis even in Atg5-deficient cells, suggesting that its effects on protein degradation extend beyond its role in autophagy inhibition(20).In this study, after treating the Huh7.5 cells with 3-methyladenine, the expression of LC3-ll was measured by flow cytometry and it decreased autophagy marker compared to the control cell.
To induce autophagy, Beclin1 transfected Huh7.5 cells showed significant increases in expression of LC3-ll, compared to control cells.