|Year : 2018 | Volume
| Issue : 12 | Page : 1141-1147
Autophagy facilitates anticancer effect of 5-fluorouracil in HCT-116 cells
Jing-wen Yang1, Qing-huai Zhang1, Tong Liu2
1 Department of Anorectal Surgery, Tianjin Union Medicine Centre, Tianjin, P.R. China
2 Department of General Surgery, Institute of General Surgery, General Hospital, Tianjin Medical University, Tianjin, P.R. China
|Date of Web Publication||11-Dec-2018|
No. 154, Anshan Road, Heping District, Tianjin 300070
Source of Support: None, Conflict of Interest: None
Aim of Study: The roles of autophagy performed in chemotherapy-induced cell death or proliferation inhibition were still in debate. In this study, we aimed to disclose the function of autophagy in chemotherapy of HCT-116 colon cells.
Materials and Methods: Pharmacological and genetic methods were applied to induce and inhibit autophagy and elucidate the roles of autophagy performed in chemotherapy-induced proliferation inhibition and apoptosis. Autophagy was assessed by microtubule-associated protein light chain 3 (LC3) expression and monodansylcadaverine (MDC) staining.
Results: After treatment with 5-fluorouracil (5-FU), HCT-116 cells showed typical autophagy as stained by MDC. Autophagy inhibitor (3-methyladenine [3-MA]) or inducer (rapamycin) was applied in combination with 5-FU, respectively. As evidenced by our data, 3-MA inhibited while rapamycin facilitated 5-FU-induced apoptosis and proliferation inhibition of HCT-116 cells. Consistently, 3-MA inhibited, while rapamycin facilitated 5-FU-induced expressions of Beclin1 and LC3B. Moreover, 3-MA inhibited while rapamycin facilitated 5-FU-induced p53 protein expression. Using genetic method, Beclin1 overexpression increased while Beclin1 knockdown decreased 5-FU-induced cell proliferation inhibition and apoptosis. Especially, Beclin1 overexpression increased while Beclin1 knockdown decreased 5-FU-induced p53 expression.
Conclusion: Our study provides both of pharmacological and genetic evidence to support that autophagy facilitates anticancer effect of the chemotherapeutic agent. The associated application of autophagy inducer with 5-FU would be beneficial for the chemotherapy in HCT-116 cancer cells.
Keywords: Apoptosis, autophagy, chemotherapy, p53
|How to cite this article:|
Yang Jw, Zhang Qh, Liu T. Autophagy facilitates anticancer effect of 5-fluorouracil in HCT-116 cells. J Can Res Ther 2018;14, Suppl S5:1141-7
| > Introduction|| |
The incidence of colorectal carcinoma is increasingly diagnosed worldwide. The normal standard of care for patients with locally advanced rectal cancer includes preoperative 5-fluorouracil (5-FU) and radiation therapy and surgical resection. Five-year survival rates vary drastically depending on pathologic response after adjuvant treatment, from 85% to 90% in patients with a pathologic complete response (pCR) to 66% in patients without pCR. Therefore, it is of particularly significant to improve the efficacy of preoperative treatment for locally advanced rectal cancer. Nevertheless, the chemotherapy was restricted by drug resistance or low sensitivity.
Autophagy is thought to be a typical type of programmed cell death and depends on lysosomal degradation of cellular organelle and protein recycling. To this end, autophagy was thought to be a double-edged sword in cell death. On the one hand, it is supposed as a type of programmed cell death. On the other hand, autophagy activation contributes to the clearance of accumulated protein or damaged organ. Increased levels of autophagy have been observed in nutrient- and oxygen-poor tumor regions as compared to highly vascularized, nutrient-enriched areas. Therefore, the roles of autophagy attract wide attention in cell survival or cell death, especially in cancer research.
Chemotherapy is still the normal therapeutic method for colorectal cancer. As previously demonstrated, autophagy functioned to promote the resistant of cancer cells to chemotherapy. In those cases, autophagy should be inhibited to increase the sensitivity of cancer cells to chemical substances. However, autophagy sometimes played an opposite roles and promoted the sensitivity of chemotherapy. In this study, we aimed to disclose the exact roles of autophagy in 5-FU-induced apoptosis of HCT-116 cells.
| > Materials and Methods|| |
Cell culture and treatments
Human colon carcinoma (HCT-116), purchased from the American Type Culture Collection (USA) was cultured in McCoy's 5A supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 1% penicillin/streptomycin (100 μg/ml; PAA Laboratories), 1% amphotericin B (250 μg/ml; PAA Laboratories). The cells were maintained in a humidified incubator (5% CO2 in air at 37°C). 5-FU was obtained from Sigma, and all compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma, USA).
The cells were incubated and cultured overnight to allow cell attachment. In this study, autophagy was induced using its inducer rapamycin or overexpression of Beclin1 while autophagy was inhibited using its inhibitor 3-methyladenine (3-MA) or knockdown expression of Beclin1. Totally, the cells were randomly divided into 5-FU, 5-FU + autophagy inducing group, 5-FU + autophagy inhibition group, and 5-Fu + z-VAD-FMK groups. After 5-FU treatment for 6, 12, and 24 h, the cell proliferation, apoptosis, and autophagy-related protein expressions were detected. Rapamycin (100 nM), 3-MA (1 μM) were co-applied with 5-FU. pcDNA3.1-Beclin1 was transfected into HCT-116 cells while pGenesil-Beclin1 small interfering siRNA was transfected to inhibit Beclin1 expression. Beclin1 expression was monitored after transfection.
Monodansylcadaverine (MDC) staining was applied to track the autophagosome. In brief, after treatment with 5-FU, the cells were fixed in paraformaldehyde and stained by MDC (50 μmol/L). The images were taken under a fluorescence microscope (excitation: 390 nm, emission: 460 nm). In each group, 100 cells were analyzed, and the numbers of autophagosome were counted.
Cells were seeded (3 × 103 cells/well) in 96-well plates in growth medium. The cells were incubated and cultured overnight to allow cell attachment. After incubated with indicated drugs, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to determine the cell proliferation. MTT solution (5 mg/ml) was added following incubation at a final concentration of 0.5 mg/ml and further incubated for 4 h. The supernatant was removed, and DMSO was added to dissolve the formed formazan crystal. The absorbance was measured by an ELISA plate reader (Bio-Rad, USA) at 570 nm wavelength.
Cells were seeded and cultured overnight to allow cell attachment. After incubated with indicated drugs, HCT-116 cells were harvested and then washed with precooled PBS before suspension in Annexin binding buffer at 1 × 106 cells/ml. Subsequently, 100 μL cell suspension was transferred to a polystyrene round-bottom tube and stained with 5 μL of Annexin V-FITC (BD Bioscience, USA) for 15 min and then with 5 μL of propidium iodide (50 μg/ml) for 2 min. Staining was performed in the dark at room temperature. After that, the cell solutions were detected by FACSCanto II flow cytometer (BD Bioscience, USA) as previously described.
Western blot analysis was performed as previously described. After incubated with indicated drugs, HCT-116 cells were harvested through scrapping. Cell lysates were collected, and protein samples were denatured at 95°C for 5 min. Subsequently, 20 μg/ml protein lysate was resolved in 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted on nitrocellulose membranes (Bio-Rad, USA). The primary antibodies used were Beclin1 (1:100, Abcam, USA), light chain 3B (LC3B) (1:100, Abcam, USA), p53 (1:100, Abcam, USA), and Actin (1:100, Abcam, USA). After incubation with the primary antibodies overnight at 4°C, the membrane was incubated with the second antibody. Enhanced chemiluminescence reagent was applied to enhance the staining. ChemiDoc™ XRS (Bio-Rad, USA) was applied to scan the blots.
Data were presented as mean ± standard deviation (SD). All the statistical analyses were performed by SPSS 17.0 (SPSS Inc., Chicago, Illinois, USA). Two-group comparison was completed by t-test. When more than two groups were compared, we used one-way or two-way ANOVA followed by Bonferroni post hoc test to determine statistical significance. P < 0.05 was considered statistically significant.
| > Results|| |
Proliferation inhibition by chemotherapy was facilitated by autophagy inducer
In our study, we first detected the autophagy of HCT-116 cells after 5-FU treatment. Obvious autophagosomes indicated by the positive staining around the nucleus were found after 5-FU treatment for 6, 12, and 24 h [Figure 1]. Moreover, our data showed that 100 μg/ml 5-FU inhibited cell proliferation (inhibition rate: 53%) after 24 h incubation [Figure 2]a. However, when co-applied with 3-MA, the inhibition rate was significantly decreased while the inhibition rate was enhanced when rapamycin was co-applied. Caspase-3 inhibitor also mitigated 5-FU-induced proliferation inhibition. 3-MA and z-VAD showed low inhibition while rapamycin induced a mild inhibition. These data suggested that 5-FU possibly causes proliferation inhibition in HCT-116 cells through facilitating autophagy. Western blotting also showed that 5-FU up-regulated Beclin1 expression [Figure 2]b.
|Figure 1: 5-fluorouracil stimulates autophagy in HCT-116 cells. (a) Representative image of monodansylcadaverine staining without 5-fluorouracil. (b) Representative image of monodansylcadaverine staining after 5-fluorouracil treatment for 6 h. (c) Representative image of monodansylcadaverine staining after 5-fluorouracil treatment for 12 h. (d) Representative image of monodansylcadaverine staining after 5-fluorouracil treatment for 24 h. (e) Quantification data of monodansylcadaverine positive cells. *P < 0.05 compared with 0 h|
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|Figure 2: 5-fluorouracil activates autophagy to inhibit the proliferation of HCT-116 cells. (a) 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay showed that 5-fluorouracil inhibited the proliferation of HCT-116 cells through promoting autophagy. (b) 5-fluorouracil increased Beclin1 expression. Two-way ANOVA, **P < 0.01 compared with 5-fluorouracil group, #P < 0.05 compared with 3-methyladenine group|
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5-fluorouracil-induced apoptosis was facilitated by autophagy inducer
We further measured the apoptosis. As shown in [Figure 3] and 5-FU elicited 20% apoptosis in HCT-116 cells. However, the apoptosis was mitigated by 3-MA, facilitated by rapamycin. 3-MA and z-VAD slightly affected apoptosis. By contrast, rapamycin induced a 10% apoptosis.
|Figure 3: 5-fluorouracil activates autophagy to promote apoptosis of HCT-116 cells. (a-h) represent the apoptosis in control, 5-fluorouracil treatment, 3-methyladenine with 5-fluorouracil, rapamycin with 5-fluorouracil, caspase-3 inhibitor with 5-fluorouracil, 3-methyladenine, rapamycin, and caspase-3 inhibitor groups. (i) Quantification data of the apoptosis. Two-way ANOVA, *P < 0.05 compared with 5-fluorouracil group, #P < 0.05 compared with 3-methyladenine group|
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5-fluorouracil-induced expression of Beclin1, light chain 3B, and p53 was facilitated by autophagy inducer
The autophagy-related protein expressions were also detected. As shown in [Figure 4]a, Beclin1 expression was significantly elevated after 5-FU incubation. 3-MA mitigated, while rapamycin facilitated the expression of Beclin1 in messenger RNA level. A similar trend was found in LC3B gene expression [Figure 4]b.
|Figure 4: 5-fluorouracil promotes p53 expression through activating autophagy. (a) Beclin1 expression, (b) light chain 3B expression, and (c) p53 expression. Two-way ANOVA, *P < 0.05 compared with control, #P < 0.05 compared with 5-fluorouracil group|
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We also detected p53 expression. As shown in [Figure 4]c, 5-FU significantly up-regulated p53, which was inhibited by 3-MA but enhanced by rapamycin.
5-fluorouracil-induced cell proliferation inhibition was facilitated by Beclin1 overexpression
As Beclin1 expression was altered after 5-FU incubation. We designed plasmids to overexpress or knock down Beclin1 expression [Figure 5]a and [Figure 5]b. Beclin1 knowdown decreased while Beclin1 overexpression increased 5-FU-induced proliferation inhibition [Figure 5]c. Beclin1 knowdown decreased while Beclin1 overexpression increased 5-FU-induced apoptosis [Figure 5]d. The autophagic activity was evaluated by detecting LC3 expression [Figure 6]. In normal HCT-116, 5-FU transferred LC3 from Type I to Type II after 6 h treatment [Figure 6]a. The transferring attained to peak at 12 h after treatment. In Beclin1 overexpression group, LC3-II and LC3-I ratio was increased after 5-FU treatment from 6 h and peaked at 48 h after treatment [Figure 6]b. In Beclin1 knockdown group, LC3-II and LC3-I ratio was decreased at the first 6 h but was upregulated from 12 h after treatment. The ratio peaked at 24 h after treatment and started to decrease 48 h after treatment [Figure 6]c.
|Figure 5: Beclin1 overexpression increased 5-fluorouracil-induced apoptosis. (a) Beclin1 plasmid increased Beclin1 expression. (b) Beclin1 silent plasmid decreased Beclin1 expression. (c) Beclin1 knowdown decreased while Beclin1 overexpression increased 5-fluorouracil-induced proliferation inhibition. (d) Beclin1 knowdown decreased while Beclin1 overexpression increased 5-fluorouracil-induced apoptosis|
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|Figure 6: 5-fluorouracil elicited light chain 3 transferring from Type I to Type II. (a) normal HCT-116; (b) Beclin1 overexpression; (c) Beclin1 knockout|
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We also detected p53 expression. Beclin1 knowdown decreased while Beclin1 overexpression increased 5-FU-induced p53 expression [Figure 7].
|Figure 7: Beclin1 knowdown decreased, while Beclin1 overexpression increased 5-fluorouracil-induced p53 expression. Two-way ANOVA, *P < 0.05 compared with 5-fluorouracil group|
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| > Discussion|| |
Autophagy plays bidirectional roles in tumor cell survival. In some cases, autophagy stimulation inhibits chemotherapeutic agents induced proliferation inhibition or cell death. However, in some other cases, autophagy induction facilitates the chemotherapeutic effects. In our study, we used both pharmacological and genetic approaches to investigate the roles of autophagy performed in cell proliferation and apoptosis of colorectal carcinoma cells. We found that autophagy induction is beneficial for the anticancer effect of 5-FU.
Autophagy is originally defined as a cellular degradation pathway, which engulfs cytoplasmic constituents, and recycles adenosine triphosphate and essential building blocks for the maintenance of cellular biosynthesis during nutrient deprivation or metabolic stress., From this point, autophagy is a cell survival mechanism. However, autophagy cell death was also found and supposed as an important programmed cell death. Therefore, autophagy is a double-edged sword in the cancer prevention. In our study, we evidenced that 5-FU elicited apoptosis in HCT-116 through autophagy activation. The activation of autophagy might promote self-consumption of the organelles and proteins, which elicits apoptosis.
Over the past decades, the selection of chemotherapeutic regimens has expanded greatly due to the development of molecular targeted therapy. Among the effective drugs, 5-FU remains to be the most popular and has been widely used as adjuvant treatment for colon cancer. However, chemoresistance and low sensitivity severely influence the chemotherapeutic effects of 5-FU. In our study, we found that 5-FU-induced autophagic responses in HCT-116 cells.
Colorectal carcinoma is one of the malignant tumors affecting human health while chemotherapy is the primary adjuvant therapy method. However, the normally used chemotherapeutic methods did not attain the ideal tumor cell killing effect. The mechanisms for chemotherapy resistance or insensitivity to chemotherapy will help to improve the chemotherapeutic effects. Interestingly, chemotherapy associated with gene therapy has been demonstrated to be a hot topic. Although autophagy stimulation was supposed to inhibit the chemotherapeutic effects, our study demonstrated opposite results showing that autophagy was beneficial for chemotherapy. The associated application of autophagy induction with chemotherapy improved the tumor killing effect and proliferative inhibition. In addition to autophagy regulated by chemical substances, we also genetically controlled autophagy by overexpressing or knocking down Beclin1 expression. Beclin1 is one of the important autophagy-related genes. Beclin1 introduces the targeted proteins to locate at preautophagosomal structure. Moreover, Beclin1 was one of the downstream of phosphoinositide 3-kinase signaling pathway, which positively regulates autophagy. Beclin1 overexpression not only increased LC3 transferring from LC3-I to LC3-II but also facilitated the proliferation inhibition effect. These data further emphasize the function of autophagy in chemotherapy effects of 5-FU. By contrast, Beclin1 knockdown decreased the autophagy and attenuated antitumor effects of 5-FU.
Recently, apoptosis is supposed as the important type of programmed cell death after radiotherapy and chemotherapy. Sensitivities to radiotherapy and/or chemotherapy are critical for the anticancer effect. In our study, we demonstrated that autophagy induction promoted apoptosis induced by 5-FU. On the one hand, rapamycin and 3-MA performed opposite effects in 5-FU-induced apoptosis of colorectal carcinoma cells. On the other hand, using genetic methods to interfere the Beclin1 expression could also effectively regulate 5-FU-induced apoptosis.
p53 gene is a tumor suppressor gene, and p53 protein stops the formation of tumors. p53 has many mechanisms of anticancer function and plays a role in apoptosis, genomic stability, and inhibition of angiogenesis. It can arrest growth by holding the cell cycle at the G1/S regulation point on DNA damage recognition. Moreover, it can initiate apoptosis if DNA damage proves to be irreparable. In our study, we found that 5-FU itself could stimulate p53 expression. Rapamycin promoted while 3-MA attenuated p53 expression. These data might suggest that autophagy induction promotes p53-dependent apoptosis in 5-FU-induced colorectal carcinoma cells. In addition, both of p53-dependent and p53-independent pathways were involved in the regulation of autophagy.
| > Conclusion|| |
In this study, we concluded that autophagy plays positive roles involved in 5-FU-induced apoptosis of HCT-116. On the one hand, 5-FU induced autophagy in HCT-116 cells. The blockage of autophagy partially inhibited the chemotherapeutic effects. On the other hand, autophagy induction facilitated the chemotherapeutic effects.
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Conflicts of interest
There are no conflicts of interest.
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