Introduction
Evidence has indicated that more than 850,000 patients are diagnosed with liver cancer each year worldwide, indicating that liver cancer is a major health issue. Hepatocellular carcinoma (HCC) accounts for approximately 90% of all primary liver cancer cases and is known as the second leading cause of cancer-related deaths worldwide (1,2). Notably, investigations have revealed that the age-standardized incidence rates (ASIRs) of liver cancer in eastern Asia are higher than those in other countries worldwide. In Taiwan, liver cancer was among the top four most common cancers in 2014, and the ASIR has decreased over the past several years (3). The development of liver cancer has been linked to a variety of risk factors, including sex, ethnicity, chronic viral hepatitis, cirrhosis, inherited metabolic disorders, alcohol abuse, tobacco use, aflatoxins, obesity, and type-2 diabetes (4,5).
The incidence of primary liver cancer is largely explained by infection with hepatitis B and C viruses, and such infections account for over 80% of liver cancer cases worldwide (6). In fact, numerous studies have demonstrated a strong correlation between chronic viral hepatitis, particularly hepatitis induced by hepatitis B and C viruses and liver cancer development (6-9). A previous study using an algorithm reported that patients who met the criteria for chronic hepatitis B virus (HBV) infection had a significantly higher incidence, ranging from 30 to 140 times, of developing HCC compared with patients without HBV (10). Another cohort study also indicated that hepatitis C virus (HCV) infection was associated with the highest incidence of HCC in patients with cirrhosis, particularly in Japan (11). More than half of HCC cases worldwide are attributable to HBV infection. Case-control and cohort studies reported that the relative risk of HCC in patients with HBV infection ranges from 5 to 49 and from 7 to 98, respectively (12,13).
Evidence has indicated that chronic HBV or HCV infection may cause liver cirrhosis, which is also the most important risk factor for liver cancer. Various studies have reported that sustained reduction in HBV/HCV replication lowers the risk of HCC in patients with HBV/HCV-associated cirrhosis (7,11). Accordingly, the primary strategy for liver cancer prevention is the elimination of viral infection by antiviral therapy (14,15). However, information concerning the anti-liver cancer effects of antiviral drugs remains obscure. Therefore, the present study investigated the effects of two anti-hepatitis virus drugs, lamivudine and ribavirin, and one anti-influenza virus drug, oseltamivir, on liver cancer cells to identify alternative methods for the treatment of liver cancer.
Materials and methods
Cell culture
Normal human liver epithelial cell line THLE-3 [CRL-11233; American Type Culture Collection (ATCC)] and two liver cancer cell lines, Huh-7 (JCRB0403; JCRB Cell Bank) and C3A [HepG2/C3A, derivative of HepG2 (ATCC HB-8065)] (CRL-10741; ATCC) were maintained following the manufacturers' instructions in bronchial epithelial cell growth medium (BEGM) (Lonza Group, Ltd.) or Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), respectively. The cell lines used in the present study were subjected to short tandem repeat (STR) profiling through the National Cheng Kung University (NCKU) Center for Genomic Medicine to confirm their authenticity. The antiviral drugs, including lamivudine (Zeffix Tablet 100 mg; GlaxoSmithKline), ribavirin (Robatrol capsule 200 mg) and oseltamivir (Tamiflu capsule 75 mg; both from Roche Diagnostics) were obtained from Changhua Christian Hospital, Taiwan.
Cell viability
To determine the survival of cells, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed. A total of 1×105 cells were cultured overnight at 37°C in each well of a 24-well plate in a cell incubator. Following incubation with different concentrations of antiviral drugs (lamivudine and ribavirin: 0, 1,000, 2,000, 3,000, 4,000 and 5,000 µM; oseltamivir: 0, 50, 100, 250, 500 and 1,000 µM), the culture medium was removed and MTT reagent (0.5 mg/ml) was added to each well and incubated for another 4 h. A total of 0.3 ml dimethyl sulfoxide (DMSO) was then added to each well of the plate and the absorbance was measured at 570 nm with a microplate reader (SpectraMax M5; Molecular Devices, LLC).
Wound healing assay
To verify the effects of oseltamivir on migration of liver cancer cells, a wound healing assay was performed. Briefly, Huh-7 and HepG2 cells were cultured in serum-free DMEM medium overnight in a 6-well plate (5×106 cells/well) until reaching 90% confluency. A sterilized 200-µl pipette tip was used to make a wound by scratching across the well surface. Following washing out the debris with fresh medium, the cells were incubated at 37°C for 24 and 48 h in the presence of various concentrations (0, 50, 100, 250, 500 and 1,000 µM) of oseltamivir and images of the wound gaps were captured at 0, 24 and 48 h using Zeiss AxioVert 200 inverted fluorescence microscope. The cell-migrated areas were calculated with Motic Images 2.0 software (Motic Incoporation, Ltd.).
Transwell migration and invasion assays
To verify the effects of oseltamivir on hepatoma cell migration and invasion, 24-well Millicell Hanging Cell Culture inserts (8-µm pore size; EMD Millipore) were used. For the invasion assay, the upper chambers were precoated with 0.4 mg/ml Matrigel (BD Biosciences) at 37°C for 24 h. Briefly, the upper chamber containing serum-free DMEM medium (2×105 cells) and various concentrations (0, 50, 100, 250, 500 and 1,000 µM) of oseltamivir, and the bottom chamber containing standard medium (DMEM with 10% FBS) was combined and incubated at 37°C for 24 and 48 h in a cell incubator. Next, the migrating cells were fixed with neutral-buffered formalin (10%) at 25°C for 2 h and then stained with 0.05% Giemsa stain at 25°C for 2 h. A total of six random fields were counted for each experiment under a light microscope at a magnification of ×200 per filter.
Flow cytometry
For flow cytometric analysis, the cells were incubated with various concentrations of oseltamivir (0, 50, 100, 250, 500 and 1,000 µM) at 37°C for 24 and 48 h. Following incubation, the 1×106 cells were harvested, washed with phosphate-buffered saline (PBS), and fixed with 70% alcohol for 16 h at -20°C. The cells were then washed with PBS and transferred into 12×75-mm tubes. A total of 10 µl of propidium iodide (PI) staining solution was added and chilled on ice in the dark. Following filtration through a 40-µm nylon screen, the cells were analyzed with a FACSCalibur analyzer (Nippon Becton Dickinson) and data analysis was performed using WinMDI 2.9 (The Scripps Research Institute, San Diego, USA).
Protein preparation and immunoblotting
The cell pellets were collected by centrifugation at 800 × g for 5 min at 4°C and suspended in 600 µl PRO-PREP™ buffer (iNtRON Biotechnology, Inc.) for lysis. The supernatant was then obtained by centrifugation at 16,600 × g for 5 min at 4°C. The concentrations of protein were measured by a modified Bradford's assay using a spectrophotometer (Hitachi U 3000; HITACHI) at 595 nm with BSA (Sigma-Aldrich; Merck KGaA) as the standard. For immunoblotting, extracted proteins (25 µg/lane) were separated by 8-12% SDS-PAGE and electrophoretically transferred to PVDF membranes (Immobilon-E, 0.45 µM; MilliporeSigma). After blocking in 5% non-fat dry milk for 1 h at 25°C, the membranes were incubated with antibodies against Apaf-1 (1:2,000; product code ab2000; Abcam), cleaved caspase-3 (1:500; product no. AB3623; Sigma-Aldrich; Merck KGaA), cleaved PARP-1 (1:500; cat. no. sc-7150; Santa Cruz Biotechnology, Inc.), LC3 (1:5,000; cat. no. NB100-2220), Beclin-1 (1:10,000; cat. no. NB110-87318), and p62/SQSTM1 (1:4,000; cat. no. NBP1-48320; all from Novus Biologicals, LLC) and β-actin (1:5,000; cat. no. MAB1501; EMD Millipore) were used to detect apoptosis and autophagy. Briefly, the PVDF membranes (Immobilon-E, 0.45 µM; MilliporeSigma) were incubated with the antibodies for 3 h at 25°C. Next, the secondary antibodies conjugated with horse-radish peroxidase (HRP) (1:5,000; cat. no. sc-2004 or sc-2005; Santa Cruz Biotechnology, Inc.) were added and incubated for 1 h at 25°C. To detect the antigen-antibody complexes, Immobilion Western HRP Chemiluminescent Substrate (EMD Millipore) and a densitometry apparatus LAS-4000 (Image Analysis Software: GE ImageQuant TL 8.1; GE Healthcare Life Sciences) were used. In addition, an autophagy inhibitor, chloroquine diphosphate (CQ; cat. no. L10382; LC3B Antibody Kit; Invitrogen; Thermo Fisher Scientific Inc.), was used to verify the participation of the autophagic mechanism to oseltamivir-induced death in Huh-7 and HepG2 cells. Following pre-treatment of Huh-7 and HepG2 cells with CQ (25 µM) for 1 h, the cells were then incubated with 1 mM oseltamivir for 24 h at 37°C.
Enzyme-linked immunosorbent assay (ELISA)
An active caspase-3 ELISA Kit (Human active caspase-3 (Ser29) SimpleStep ELISA kit; product code ab181418; Abcam) was used to measure active caspase-3 according to the manufacturer's protocol.
Immunofluorescence staining
An LC3B Antibody kit (cat. no. L10382; Invitrogen; Thermo Fisher Scientific Inc.) was used for autophagy detection according to the manufacturer's protocol. Briefly, the 1×106 cells were seeded on Millicell EZ SLIDE 8-well glass slides and maintained with fresh DMEM with 10% FBS medium containing various doses of oseltamivir (0, 50, 100, 250, 500 and 1,000 µM) for 24 h. Next, the cells were blocked in 2% BSA buffer at 25°C for 1 h and then washed with 1X PBS and fixed with 4% paraformaldehyde at 25°C for 15 min. Subsequently, the cells were permeabilized with 0.3% Triton X-100 at 25°C for 15 min, followed by reacting with antibodies against LC3-B (0.5 µg/ml). Following incubation with Alexa Fluor® 488 goat anti-rabbit IgG (H+L) antibodies (1:500; cat. no. A21206, Invitrogen; Thermo Fisher Scientific Inc.) at 25°C for 1 h, one drop ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific Inc.) was used to mount the coverslips at 25°C for 1 min. The cells were observed under a ZEISS AXioskop2 fluorescence microscope (Carl Zeiss Microscopy, LLC).
Xenograft study
A total of 15 female athymic nude mice (5-weeks old; weight 15-17 g) were acquired from National Center for Experimental Animals of Taiwan and kept in specific-pathogen-free (SPF) facilities with a 12-h light/dark cycle and a relative humidity in an airconditioned room of 55%. Animals were allowed free access to sterilized water and chow (Lab Diet 5001; PMI Nutrition International Inc.) at Chung Shan Medical University (Taichung, Taiwan). Study protocols were authorized by the Institutional Animal Care and Use Committee of Chung Shan Medical University, Taiwan, R.O.C. (IACUC approval no. 2542). The study was carried out in compliance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). A total of 5×106 Huh-7 cells in PBS were hypodermically injected into the flank of mice at the age of 6-weeks old. The doses of oseltamivir used in the xenograft study were based on a previous study (16). When the tumor volumes reached ~100 mm3, the mice were randomly divided into three groups including control, low dose- and high-dose groups and were daily intraperitoneally injected with PBS, 15 and 60 mg/kg oseltamivir, respectively. The tumor diameters and volume were measured every two days using a caliper. All the mice were sacrificed (performed on August 3, 2021) when the tumor volumes of the mice from the control group reached ~2,000 mm3. Inhalation of carbon dioxide (CO2) was used for mice euthanasia. The flow rate of CO2 was 50% of the chamber volume/min. Following visual confirmation of respiratory cessation of the mice, the CO2 flow was maintained for 1 min to ensure the death of mice.
Statistical analysis
GraphPad Prism 5 software (GraphPad Software, Inc.) was used to calculate the significant differences among groups. For the MTT, wound healing, and Transwell migration assays, as well as ELISA and immunoblotting, two-way ANOVA with Bonferroni's post hoc test for multiple comparisons was performed to calculate the effects of drug treatment. P<0.05 was considered to indicate a statistically significant difference. All values are presented as the mean ± SEM.
Results
Effects of lamivudine, ribavirin and oseltamivir on the proliferation, migration and invasion of liver cancer cells
To assess the effects of antiviral drugs, including lamivudine, ribavirin, and oseltamivir, on liver cancer, Huh-7 and HepG2 cells were first treated with various concentrations of lamivudine, ribavirin, and oseltamivir. THLE-3 cells were used as normal control cells. Although lamivudine caused a statistically significant difference in cell viability compared with the control treatment (0 µM), lamivudine had little effect on the survival of THLE-3, Huh-7, and HepG2 cells (Fig. 1). All three cell lines exhibited significantly decreased viability in the presence of ribavirin and oseltamivir in a dose-dependent manner at 24 (Fig. 1A) and 48 h (Fig. 1B). Notably, Huh-7 and HepG2 cells exhibited significantly lower viability in the presence of 250 and 500 µM oseltamivir at 48 h compared with THLE-3 cells (Fig. 1B). Since more aggravated clinical adverse effects have been reported with ribavirin than oseltamivir, the following studies focused on the effects of oseltamivir on liver cancer cells. To further investigate the effects of oseltamivir on the migration and invasion of Huh-7 and HepG2 cells, wound healing and Transwell assays were performed. Significantly decreased migrating areas were detected in both Huh-7 and HepG2 cells treated with oseltamivir for 24 and 48 h (Figs. 2A and 3A). Significantly decreased migration and invasion were observed in both Huh-7 and HepG2 cells that were treated with oseltamivir for 24 h (Figs. 2B and 3B).
Oseltamivir induces autophagy but not apoptosis in Huh-7 cells
To verify whether apoptosis and autophagy are involved in oseltamivir-induced Huh-7 cell death, flow cytometry, immunoblotting, immunofluorescence, and caspase-3 ELISAs were performed. A slightly increased proportion of Huh-7 cells in the sub-G1 phase were observed following treatment with different concentrations of oseltamivir for 24 and 48 h (Fig. 4A-C). No statistically significant differences in the protein expression of Apaf-1, cleaved caspase-3 and cleaved PARP-1 (Fig. 5A-D) or the activity of caspase-3 (Fig. 5E) were observed in the presence of oseltamivir for 24 h. Notable protein expression of LC3-II was detected in Huh-7 cells treated with 250, 500, and 1,000 µM oseltamivir for 24 h (Fig. 6). In addition, a significantly higher ratio of LC3-II/LC3-I and increased protein expression of Beclin-1 was observed in Huh-7 cells in a dose-dependent manner (Fig. 7A and B). Conversely, significantly decreased protein expression of p62 was detected in Huh-7 cells treated with 250, 500, and 1,000 µM oseltamivir for 24 h (Fig. 7C).
Oseltamivir induces both apoptosis and autophagy in HepG2 cells
To verify whether apoptosis and autophagy are involved in oseltamivir-induced HepG2 cell death, flow cytometry, immunoblotting, immunofluorescence, and caspase-3 ELISAs were performed. Significantly increased proportion of HepG2 cells in the sub-G1 phase were observed following treatment with 500 and 1,000 µM oseltamivir for 24 h and with 100, 250, 500, and 1,000 µM for 48 h (Fig. 8A-C). Accordingly, significantly increased protein expression of Apaf-1, cleaved caspase-3, and cleaved PARP-1 was detected in HepG2 cells treated with oseltamivir for 24 h (Fig. 9A-D). Additionally, significantly increased caspase-3 activity was observed following treatment with 250, 500, and 1,000 µM oseltamivir for 24 h (Fig. 9E). A notably higher amount of LC3-II protein was observed in HepG2 cells treated with 250, 500, and 1,000 µM oseltamivir for 24 h (Fig. 10). A significantly higher ratio of LC3-II/LC3-I and increased protein expression of Beclin-1 were detected in Huh-7 cells in a dose-dependent manner, whereas significantly decreased protein expression of p62 was detected following treatment with 50, 100, 250, 500 and 1,000 µM oseltamivir for 24 h (Fig. 11). In addition, an autophagy inhibitor, CQ, was used to verify the participation of the autophagic mechanism to oseltamivir-induced death in Huh-7 and HepG2 cells (Fig. 12). Following pre-treatment of Huh-7 and HepG2 cells with CQ (25 µM) for 1 h, the cells were then incubated with 1 mM oseltamivir for 24 h. As anticipated, reduced p62 and increased LC3-II protein levels were observed in both Huh-7 and HepG2 cells that were treated with 1 mM oseltamivir compared with the cells cultured in absence of both CQ and oseltamivir. Inhibition of lysosomal degradation by pre-treatment with CQ prevented the decomposition of LC3-II and p62 and resulted in accumulation of LC3-II and p62 (Fig. 12).
Oseltamivir inhibits the growth of xenografted Huh-7 cells in nude mice
To assess the effects of oseltamivir in vivo, xenografted tumors were generated by hypodermic injection of 5×106 Huh-7 cells into athymic nude mice. When the tumor volume was ~100 mm3, the animals were treated daily with PBS (control group), 15 mg/kg oseltamivir (low-dose group) and 60 mg/kg oseltamivir (high-dose group), respectively. A significantly smaller mean tumor volume was detected in the mice treated with 15 mg/kg or 60 mg/kg oseltamivir as compared with the mice treated with PBS from day-4 (Fig. 13A). Notably, a significantly smaller mean tumor volume was detected in the mice treated with 60 mg/kg oseltamivir as compared with the mice treated with 15 mg/kg from day-10 (Fig. 13A). Fig. 13B reveals the representative xenografted tumors retrieved at the end of the experiments.
Discussion
Since hepatitis virus infection has been strongly associated with the occurrence of liver cirrhosis and HCC, the traditional therapeutic strategy for liver cancer is to eliminate the hepatitis virus (7,17). In addition to drugs that target hepatitis viruses, various drugs against other viruses have been used for the treatment of liver cancer. However, information concerning the effects and related mechanisms of these antiviral drugs remains limited. In the present study, it was revealed that oseltamivir, an anti-influenza virus drug, significantly inhibited the growth and migration of Huh-7 and HepG2 cells. Oseltamivir also exerted differential effects on these liver cancer cell lines by inducing apoptosis and autophagy alone or in combination.
Evidence has demonstrated dual roles of autophagy, namely, both tumor inhibitory and tumor promoting roles, in cancers. A variety of studies have revealed that autophagy promotes cancer cell death in early phases and survival in later phases (18-20). Autophagy may provide energy for excessive cancer cell proliferation or lead to the insufficient availability of nutrients for cancer cells by disrupting energy homeostasis, thus causing cell death (21). A previous study reported that quinacrine-induced autophagy in ovarian cancer cells leads to excessive autophagic flux and promotes cell death (22). Another study indicated that dihydroartemisinin (DHA)-37, an analog of DHA, exhibits significant anticancer activity against A549 cells by triggering excessive autophagic cell death (23). A recent study also revealed that Dendrobium officinale polysaccharide significantly inhibits CT26 cell proliferation by inducing mitochondrial dysfunction and excessive autophagy (24). These findings suggested that excessive amounts of autophagy could be a potential strategy for causing cancer cell death. Accordingly, the present study reported, for the first time to the best of our knowledge, that oseltamivir induces excessive autophagic cell death in both Huh-7 cells and HepG2 cells, providing an alternative method for the treatment of liver cancer.
An alternative approach based on the use of non-oncological drugs for cancer treatment has attracted considerable attention in recent decades (25,26). Various types of non-oncological medicines, including antiviral drugs, have yielded promising in vitro results and are already being assessed in clinical trials (27). In the present study, it was revealed that antiviral drugs, including lamivudine, ribavirin, and oseltamivir, exerted significant inhibitory effects on the proliferation of Huh-7 and HepG2 cells. However, the concentrations of lamivudine required to inhibit the proliferation of these liver cancer cell lines were markedly higher than those of both ribavirin and oseltamivir. In addition, ribavirin usually needs to be combined with interferon in clinical treatment and requires a higher dose and longer treatment time, which lead to more serious side effects (28). Notably, the dose of oseltamivir that was significantly effective in inhibiting proliferation was markedly lower than that of ribavirin. Thus, the dose of this medication could be reduced while still achieving therapeutic efficacy, which could prevent adverse side effects, drug diversion, poisoning, and waste treatment (29). In addition to the significant inhibitory effects of oseltamivir on Huh-7 and HepG2 cells, these findings suggested additional potential of oseltamivir in the treatment of liver cancer in the clinic.
Therapeutic strategies targeting apoptosis (30,31) and autophagy (32,33) have long been used for cancer treatment. An interesting finding in the present study was the differential effects of oseltamivir on Huh-7 and HepG2 cells. Oseltamivir induced both autophagy and apoptosis in HepG2 cells but induced only autophagy in Huh-7 cells. This phenomenon may be attributed to the different characteristics of the two cell lines, particularly the mutation of p53 in Huh-7 cells (33). A previous genetic study reported that HepG2 cells have a N-ras mutation at codon 61, and Huh-7 cells have a missense mutation in the p53 gene at codon 220, resulting in an amino acid change of cysteine for tyrosine (34). p53 is one of the most well investigated and most frequently mutated genes in various human cancers (35). A previous study has indicated that the p53 gene plays an essential role in limiting cancer formation by modulating metabolism, reactive oxygen species production, and noncoding RNA expression and by enhancing autophagy or ferroptosis (36). Indeed, mutations in the p53 gene have been demonstrated to be involved in cancer formation and progression and are present in ~50% of aggressive tumors (35). Notably, two clinical studies indicated that the presence of missense p53 mutations is significantly associated with breast cancer specificity and overall mortality (37,38). Interestingly, p53 was also found to play a key role on controlling the switch between autophagy and apoptosis. An in vitro model reported that sodium selenite switched protective autophagy to apoptosis in both a p53-wild type (NB4 cells) and p53-mutant cell model (Jurkat cells) (39,40). These findings may provide possible explanations of the difference in oseltamivir-induced death between Huh-7 and HepG2 cells. However, further investigations such as xenografted HepG2 experiments and underlying signaling analysis are required to identify the precise mechanism of oseltamivir-induced cell apoptosis and/or autophagy in Huh-7 and HepG2 cells.
In addition, the role of p62 in autophagy and apoptosis has received a great amount of attention in recent years. The scaffold protein p62, namely sequestosome 1 (SQSTM1), is well-known as a critical regulator in the autophagic process by directly binding LC3 for autophagosome generation (41,42). Evidence has indicated that p62/SQSTM1 mediated a variety of essential cellular processes, including autophagy and apoptosis, through its different domains (42,43). In fact, studies have demonstrated that p62 binds LC3 by the LC3-interacting region (LIR) within p62 and promotes the formation of autophagosomes (44,45). Additionally, p62 has also been reported to induce apoptosis through the caspase-8 activation at the autophagosomal membrane (46). In the presence of culin3, caspase-8 was modified and interacted with p62 and TRAF6, which leads to the activation of caspase-8 downstream caspase and apoptosis (47,48). These findings indicated a dual role of p62 in autophagy and apoptosis and may provide another possible rationale for explaining the difference of oseltamivir-induced autophagy/apoptosis in Huh-7 and HepG2 cells. Definitely, further experiments are merited to investigate the precise role of p62 on oseltamivir-induced cell death in Huh-7 and HepG2 cells.
Neuraminidase (NEU) is the enzyme expressed on the surface of influenza viruses, and it facilitates the release and trafficking of influenza viruses within the respiratory tract (39). Oseltamivir (Tamiflu) is an FDA-approved NEU inhibitor for the prevention and treatment of influenza A and B infections (49,50). Certain investigations have been conducted on the alternative use of oseltamivir in cancer treatment. A previous study on alternative treatments for pancreatic cancer indicated that oseltamivir inhibits the activity of NEU-1 (Sialidase) and suppresses intrinsic signaling that promotes human pancreatic cancer (PANC1) cell survival (51). In addition, oseltamivir also overcame the chemoresistance of PANC1 cells to cisplatin and gemcitabine alone or in combination by reversing changes in E-cadherin and N-cadherin expression (41). A recent study indicated an association between NEU-1 and HCC (51). Notably increased mRNA and protein expression of NEU-1 was observed in HBV-related HCC tissues, and this increased expression was caused by the binding of the HBV core protein to NF-KB on the NEU-1 promoter, which led to downstream oncogenic signaling and epithelial-mesenchymal transition (EMT) in HCC cells, including Huh-7 and HepG2 cells (52). These findings indicated the involvement of NEU in the carcinogenesis of HCC. Consistently, the present study was the first to report the anti-liver cancer activity of oseltamivir by inducing apoptosis in Huh-7 cells and inducing both apoptosis and autophagy in HepG2 cells. However, the roles of NEU and intrinsic signaling in oseltamivir-induced Huh-7 and HepG2 cell death remain unknown and warrant further investigation in order to elucidate the precise mechanism underlying the oseltamivir-induced cell death of Huh-7 and HepG2 cells. In summary, the present study provided novel findings about the use of non-oncological drugs for the treatment of liver cancer and suggests the use of oseltamivir as an alternative approach for liver cancer treatment.