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Tenacissoside H Induces Autophagy and Radiosensitivity of Hepatocellular Carcinoma Cells by PI3K/Akt/mTOR Signaling Pathway

Tenacissoside H (TEH), which has anti-inflammatory and anti-tumor effects, is a major active ingredient extracted from the stem of Marsdenia tenacissima. However, the effect of TEH on hepatocellular carcinoma (HCC) as well as the underlying mechanisms are still indistinct. Presently, HCC cells (including Huh-7 and HepG2) were dealt with different concentrations of TEH. The proliferation and apoptosis of HCC cells were determined via Cell Counting Kit-8 (CCK8) assay and flow cytometry. In addition, Western blot was conducted to evaluate the expressions of autophagy—and apoptosis-related proteins. Tissue immunofluorescence was carried out to evaluate LC3B expression in the tumor tissues. The data showed that TEH suppressed the growth of HCC cells in a concentration-dependent manner. Besides, TEH enhanced radiosensitivity and promoted the apoptosis of HCC cells. Moreover, the mRNA and protein levels of autophagy-related genes (LC3-II/LC2-I, ATG5, Beclin-1) were significantly promoted by TEH. Mechanistically, TEH attenuated the activation of PI3K/Akt/mTOR signaling pathway. However, inhibition of PI3 K pathway abolished the anti-tumor effects of TEH in HCC cells. Collectively, this study suggested that TEH increases the radiosensitivity of HCC cells via inducing autophagy and apoptosis through downregulating PI3K/Akt/mTOR signaling pathway.


Introduction
Hepatocellular carcinoma (HCC) ranks the sixth most common malignant tumors, and has a mortality rate ranked third worldwide. 1 Moreover, HCC often proceedes fast, and the 5 year survival rate of HCC patients is less than 30%. 2 Surgical treatment is the preferred method for early HCC. In addition, chemotherapy and radiotherapy are also vital therapeutic methods for patients with advanced HCC, but the patients' life quality after traditional treatments remains unsatisfactory. 3 Even worse, during radiotherapy, certain tumor cells gradually adapt to the radiation and develop radiation resistance, leading to treatment failure (recurrence or metastasis). [4][5] Therefore, it is necessary to study the mechanism of radiotherapy resistance and enhance the radiosensitivity of HCC.
Autophagy, a highly conserved process of material turnover in cells, exists in eukaryotes. Autophagy can fulfill the needs of cell metabolism, renew organelles and maintain cell homeostasis, but aberrant autophagy may lead to the emergence of cancer cells, such as enhanced autophagy in breast cancer 6 and esophageal cancer. 7 Under normal conditions, autophagy is maintained at a low level, but in case of stress, such as ionizing radiation, autophagy is activated to protect cells from environmental damage, thus generating radiation resistance. 8 Moreover, the phosphoinositol 3-kinase (PI3 K)/ protein kinase B (Akt)/rapamycin (mTOR) signaling pathway acts as a crucial regulator of autophagy and takes part in the occurrence development of many tumors. For example, resveratrol, a natural polyphenol plant antitoxin, promotes autophagy by restraining the PI3K/Akt/mTOR signaling pathway, thereby suppressing the activity and migration of melanoma cells. 9 Besides, curcumin can induce the apoptosis and autophagy of human lung cancer cell A549 by inhibiting the PI3K/Akt/ mTOR pathway, thus effectively suppressing tumor cells' growth. 10 Those researches all prove that targeting PI3K/Akt/ mTOR pathway restrains the progression of malignant tumors by modulating autophagy.
Tenacissoside H (molecular formula: C 42 H 66 O 14 , TEH) is a Chinese medicine monomer extracted from the dried stem of Marsdenia tenacissima. Emerging evidence has proved that TEH has anti-inflammatory 11 and anti-tumor 12 effects. For instance, TEH suppresses the proliferation, and arrests the cell cycle in S phase of esophageal cancer by inhibiting the PI3K/ Akt pathway. 13 However, the mechanism of TEH in HCC needs further exploration.
Presently, it was discovered that TEH could not only inhibit HCC cells' proliferation and promote cell apoptosis, but also improve tumor cells' sensitivity to radiotherapy. Moreover, TEH markedly repressed the activation of PI3K/Akt/mTOR pathway. Therefore, we hypothesized that TEH could affect the development of HCC through the PI3K/Akt/mTOR pathway via inducing autophagy, which is possible to be a potential therapeutic drug for HCC.

CCK8 Assay
Huh-7 and HepG2 cells (2 Â 10 3 ) were inoculated in 96-well plates with 100 mL cell suspension per well and cultured in an incubator at 37 C with 5% CO 2 saturated humidity. After the cells were adherent to the wall, TEH at different concentrations (0, 2.5, 5, 10 and 20 mM) was added. After continuous culturing for 0, 24, 48, and 72 hours, we added 10 mL CCK8 solution (Beyotime Biotechnology, Shanghai, China) to each well, gently shook well and incubated the culture plate in the incubator for 2 hours. Microplate Reader determined the absorbance value (A) of each well at 450 nm. Cell survival rate (%) ¼ (experimental group A/ control group A) Â100%.

Radiosensitivity Test
The radiosensitivity experiment consists of the blank control group (DMSO) and the TEH group with different concentrations (0, 5, 10, 20 mM). Huh-7 and HepG2 cells were cultured for 48 h. The cells were digested into a single-cell suspension, which was divided into 2-ml culture bottles and exposed to different doses. Irradiation conditions: 8 MV linear accelerator (varianclinicl8, USA), with the addition of 2 cm equivalent tissue filling material, irradiated 0, 2, 4, 6 and 8 Gy/min, respectively. To analyze the radiosensitivity of the cells, the cell proliferation was detected via CCK8 assay after irradiation. Huh-7 and HepG2 cells were inoculated in 96-well plates (2 Â 10 3 cells, 100 mL cell suspension per well). After 24 hours culture, the cells were treated in accordance with the previous grouping for 48 h. With the addition of 10 mL CCK8 reagent (Beyotime Biotechnology, Shanghai, China) in each cell well, the cells underwent further incubation (37 C, 4 h). Finally, a Microplate Reader was used to determine the absorbance value (A value) of each group at 570 nm.

Flow Cytometry
According to the manufacturer's instructions, AnnexinV/7-AADA apoptosis Detection Kit (Southern Biotechnology, Birmingham, Al, USA) was applied to examine the degree of cell apoptosis. The treated cells were trypsinized, centrifuged, washed twice with cold PBS and resuspended using binding buffer till a final concentration of 1.5 Â10 4 cell/mL. Then, they went through double-staining with AnnexinV and 7-AAD, and incubated 15 minutes in darkness (15-25 C, room temperature). Finally, we performed the flow cytometry to analyze the stained cells with a FACS Calibur Flow cytometry (BD Biosciences, SanJose, CA, USA). We duplicated the experiment 3 times. In the subsequent experiments, after adherent culture, the cells were treated with TEH group (5 mM), gamma-ray (4 Gy) and PI3 K inhibitor (1 mM PI-103), and the rest steps were the same as the above.

Xenograft Tumor Experiment
Forty nude female BALB/c mice (6 8 weeks old, 20 + 2 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China). Human HepG2 cells were used to test the effect of TEH on HCC cell growth in vivo. The xenograft model was established by subcutaneous injection of 1 Â 10 7 cells/100 mL cell suspension. When the tumors were formed with a volume of 75 100 mm 3 , the nude mice were randomly divided into 4 groups, including control group, TEH group (5, 10, 20 mmol/kg body weight). PBS (100 ml) were used as negative control in the veh group. TEH was administered via intraperitoneal injection 3 times a week for 4 weeks. During the treatment, the weights of the mice and the volume of the tumors were measured every 4 days. The volume of the tumors was calculated according to the following formula: V ¼ 0.5Âlength Âwidth 2 . Finally, the mice were sacrificed at the fourth week of xenograft tumor experiment. Then the tumors were carefully resected and the weight of the tumors were counted. All the animal procedures were approved by the Animal Care and Use Committee of Peking University Shenzhen Hospital.

Statistical Analysis
We took SPSS17.0 statistical software (SPSS Inc, Chicago, IL, USA) for data analysis, t-test for comparison of the mean between 2 sample groups and one-way analysis of variance (ANOVA) for the difference between multiple groups. The measurement data were expressed as mean + standard deviation (x + s). We considered P < 0.05 as statistically valuable.

TEH Inhibited the Proliferation of HCC Cells
For the purpose of detecting the function of TEH in the growth of HCC cells, different concentrations of TEH (0, 2.5, 5, 10, 20 mM) were used to treat Huh-7 and HepG2 cells for 0, 24, 48 and 72 hours, and cell proliferation and apoptosis were detected via CCK8 assay and flow cytometry. The data illustrated that TEH markedly repressed the proliferation of HCC cells with a dose-dependent manner ( Figure 1A and B). In addition, the apoptosis of HCC cells was significantly accelerated with the increase of TEH's doses ( Figure 1C and D). Therefore, the results implied that TEH limits the growth of HCC cells in a dose-dependent manner and promotes cell apoptosis.

TEH Repressed the Growth of HCC Cells In Vivo and Induced Autophagy
To further confirm the effects of TEH on HCC cells, we performed in vivo experiment. Our data showed that compared with veh group the volume and weight of the xenograft tumors in the TEH group were significantly reduced (Figure 2A-C). Moreover, with the increase of TEH's concentration, the inhibitive effect on tumor growth became more significant ( Figure  2A-C). By detecting the apoptosis-realted markers (including Bax and Bcl2), we found that TEH promoted Bax and inhibited Bcl2 expressions in a dose-dependent manner (in comparison to veh group, Figure 2D). Further, tissue immunofluorescence was conducted to evaluate the autophagy marker LC3B in the tumor tissues. It was found that TEH markedly promoted LC3B expression in the tumor tissues ( Figure 2E-F). Therefore, the in-vivo experiment further confirmed that TEH has an antitumor role in HCC cells via enhancing apoptosis and autophagy.

TEH Enhanced the Radiosensitivity of HCC Cells
To verify TEH's effect on the radiosensitivity of HCC cells, different concentrations (0, 5, 10, 20 mM) of TEH were added to Huh-7 and HepG2 cells and exposed to different doses (0, 2, 4, 6, 8 Gy) of ionizing radiation. The results of radiosensitivity experiment revealed that, the cell survival rate significantly decreased with the increase of TEH concentration and radiation dose, and the higher the concentration of TEH, the lower the cell survival rate, compared with the veh group (0 mM TEH) (P < 0.05, Figure 3A-B). The statistics suggest that TEH enhances the sensitivity of HCC cells to ionizing radiation.

TEH Promoted the Apoptosis of HCC Cells Induced by Ionizing Radiation
To explore TEH's effect on the apoptosis of irradiated HCC cells, Huh-7 and HepG2 cells were dealt with TEH (5 mM) and gamma-ray (4 Gy). Flow cytometry results represented that, compared with the control group (0GyþDMSO), the cell apoptosis rates after TEH intervention alone and after radiation intervention alone were increased. Interestingly, the combination of TEH with ionizing radiation prominently enhanced the apoptotic level of HCC cells (compared with 0Gyþ TEH group or 4 GyþDMSO group) ( Figure 4A and B). Furthermore, the Western bolt was applied to detect apoptosis-related proteins. The results illustrated that, compared with the control group, the pro-apoptotic protein Bax was upregualted and the antiapoptotic protein Bcl2 was downregulated in 0Gyþ TEH group or 4 GyþDMSO group. Interestingly, the combination of TEH and radiation led to higher level of Bax and lower level of Bcl2 (compared with 0Gyþ TEH group or 4 GyþDMSO group) (P < 0.05, Figure 4C). These findings indicated that combining radiation and TEH aggravated the apoptosis HCC cells.

TEH Promoted Autophagy and Inhibited the PI3K/Akt/ mTOR Signaling Pathway in HCC Cells
To approach the mechanism of TEH on HCC cells, Western bolt was performed to evaluate the expression of autophagyrelated proteins LC3-I/II, AGT5 and Beclin1 in HCC cells. The results displayed that, compared with the control group (0GyþDMSO), autophagy-related proteins LC3-II/I, AGT5 and Beclin1 were up-regulated after TEH intervention alone. At the same time, LC3-II/I, ATG5 and Beclin1 expressions were up-regulated after radiation treatment ( Figure 5A). Interestingly, the combination of TEH and radiation treatment promoted autophagy (compared with 0Gyþ TEH group or 4 GyþDMSO group) ( Figure 5A). Further, Western bolt was employed to measure the effect of TEH on the PI3K/Akt/mTOR pathway. The result showed that compared with the control group, the expressions of p-PI3 K, p-Akt and p-mTOR were both down-regulated in the 0Gyþ TEH group and 4 GyþDMSO group. However, the addition of TEH significantly inhibited the activation of PI3K/Akt/mTOR pathway (P < 0.05, Figure 5B). These data suggested that TEH suppresses the PI3K/Akt/mTOR signaling pathway by activating autophagy of HCC cells.

Inhibiting PI3K/Akt/mTOR Signaling Pathway Dampened TEH-Induced Effects in HCC Cells
To further study whether TEH could affect the progression of HCC via PI3 K pathway, we gave PI-103 (1 mM) to intervene the PI3 K pathway activation in Huh-7 and HepG2 cells. CCK8 assay was taken to detect cell proliferation, and flow cytometry was performed to examine cell apoptosis. The results demonstrated that, compared with the control group (4GyþDMSO), the cell proliferation in the 4GyþTEH group and the 4Gyþ PI-103 group were remarkably decreased, and the cell apoptosis considerably increased ( Figure 6A-C). However, the proliferative and apoptotic levels in the 4GyþPI-103þTEH group were not   significantly changed in contrast to those in the 4GyþPI-103 group ( Figure 6A-C). Moreover, Western blot was used to detect the expressions of autophagy-related proteins and PI3K/Akt/mTOR pathway. The results indicated that compared with the control group, autophagy-related proteins LC3-II/I, AGT5 and Beclin1 were notably up-regulated with the addition of PI-103, while the expressions of p-PI3 K, p-Akt and p-mTOR were notably suppressed ( Figure 6D and E). However, the combination of TEH and PI-103 had no significant effects on the autophagy and PI3K/Akt/mTOR pathway expression (compared with 4GyþPI-103 group). Taken together, the above studies indicated that TEH reduced the proliferation of HCC cells, promoted cell apoptosis, promoted cell autophagy and radiosensitivity dependently via inhibiting the PI3K/AKt/mTOR pathway.

Discussion
In this study, we investigated the biofunctions of TEH in HCC development. The results revealed that TEH inhibits HCC cell growth by aggravating apoptosis and autophagy dependently through repressing the PI3K/AKt/mTOR pathway.
The development of HCC is rooted in uncontrolled cancer cell growth and metastasis. Inhibiting the proliferation of HCC cells and inducing cell apoptosis are the main strategies for its treatment. 14-15 Radiotherapy causes radioactive damage when rays directly act on biomolecules, indirectly damages DNA by producing free radicals to interact with biological macromolecules, blocks cell division and proliferation, and causes irreversible damage in cells, which is the first choice for treatment of locally advanced HCC. 16 However, radiation resistance is the major challenge during radiotherapy. After long-term radiation irradiation, tumors show radiation resistance similar to chemotherapy drug tolerance, which reduces the sensitivity of the tumor to radiation and causes tumor metastasis or recurrence. 17 Overcoming or eliminating radiation resistance during radiotherapy is crucial to improving the survival rate of HCC patients.
In recent years, abundant literatures have manifested that some plant extracts make great contribution to the inhibition of HCC development. For instance, the licorice roots extract can induce apoptosis and cell cycle arrest of HCC cells via modulating tumor suppressor miRNAs, inhibiting HIF1a, PI3 K, C-Myc and activating AMPK and p53 pathway. 18 As another example, Oleanolic acid attenuates the development of HCC by inducing autophagy and promoting apoptosis. 19 TEH, along with the other 2 ingredients (tenacissoside I and G), is extracted form Marsdenia tenacissima and has been found with anti-inflammatory and anti-tumor effects. 20 Interestingly, a recent study proved that the Marsdenia tenacissima extract (MTE) promotes gefitinib accumulation in lung cancer tissues via fasciliating the activities of ABCG2, which is the primary drug transporter for gefitinib. 21 Our study found that with the increase of TEH concentration, the proliferative ability of HCC cells decreased and cell apoptosis increased. Additionally, TEH reduced the growth of HCC cells under irradiation treatment, indicating that TEH is a promising anti-tumor drug in HCC.
Autophagy, a self-digestion phenomenon in eukaryotes, is a double-edged sword that can promote or inhibit cell apoptosis.
In the general state, autophagy is maintained at a low level. When stimulated by radiation, the autophagy level is sharply up-regulated, and even type II programed cell death occurs, resulting in radiotherapy resistance. 22 However, several studies have proven that autophagy may reduce chem-or radio-resistance. [23][24] For example, inhibition of autophagy in esophageal carcinoma can improve the radiosensitivity of cancer cells. 25 Similarly, 3-methyladenine (3-MA), an autophagy inhibitor, sensitizes radio-resistant HepG2 cells to radiationinduced cytotoxicity, 26 proving that modulating autophagy is a powerful method in treating HCC. Here, our data showed that both TEH and irradiation enhanced the autophagy of HCC cells. More interestingly, the combination of the 2 treatments significantly enhances the autophagic level, suggesting that TEH facilitating irradiation induced cell growth inhibition by modulating autophagic cell death. Figure 6. TEH inhibited the progression of HCC through PI3K/Akt/mTOR signaling pathway. Huh-7 and HepG2 cells were treated with TEH (5 mM) and/or PI-103 (1 mM) and then subjected to irradiation (4 Gy). (A-B) CCK8 experiment was carried out to examine the proliferation of Huh-7 and HepG2 cells. (C) The apoptosis rate of Huh-7 and HepG2 cells was evaluated via flow cytometry. (D-E) Western bolt was applied to measure autophagy-related proteins and PI3 K / Akt / mTOR pathway expression. *P < 0.05, **P < 0.01, ***P < 0.001 (vs.4 Gy þ DMSO group); ns P > 0.05 (vs.4 Gy þ PI-103 group).