PI3K/AKT1 Signaling Pathway Mediates Sinomenine-Induced Hepatocellular Carcinoma Cells Apoptosis: An in Vitro and in Vivo Study

Abstract

Hepatocellular carcinoma (HCC) is one of the most frequent cancers. Sinomenine (SIN) is a compound derived from Sinomenium acutum. Our previous investigations have found that SIN inhibited protein kinase B (AKT) signaling to induce autophagic death of tumor cells. However, whether inhibition of this pathway by SIN could impact the proliferation of HCC cells is unknown. Thus, we applied SIN to SK-Hep-1 cells and used cell counting kit 8 (CCK8), lactate dehydrogenase (LDH), colony formation and 5-ethynyl-20-deoxyuridine (EdU) incorporation experiments to detect cell viability. Then, staining with annexin V/propidium iodide (PI) coupled with terminal deoxynucleotidyl transferase-mediated biotinylated uridine 5′-triphosphate (UTP) nick end labeling (TUNEL) staining were utilized to monitor apoptosis. Changes in cell mitochondrial membrane capacity were explored via 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, whilst Western blot or immunohistochemistry was applied to evaluate the expression levels of key proteins, consisting of Cleaved Caspase 3, AKT1, B-cell leukemia/lymphoma 2 (BCL-2), phosphatidylinositol 3-kinase (PI3K) p85α, and Cleaved Caspase 9 etc. The Balb/c nude mice were utilized to establish HCC xenograft tumor model, administered by SIN. After treatments, the tumor volume along with weight were measured. The results illustrated that SIN suppressed SK-Hep-1 HCC cells’ proliferation, enhanced the collapse of potential of the mitochondrial membrane, triggered cell apoptosis, down-regulated PI3K p85α, AKT1, BCL-2, Pro-Caspase 9, Pro-Caspase 3 expressions, and up-regulated Cleaved Caspase 9 and Cleaved Caspase 3 expressions in vitro and in vivo. Meanwhile, SIN reduced the tumor volume along with weight of mice. In addition, insulin-like growth factor-1 (IGF-1), a powerful activator of the PI3K/AKT pathway, could reverse the high apoptosis of SK-Hep-1 HCC cells induced by SIN. Overall, inhibition of PI3K/AKT1 signaling cascade by SIN induced HCC cells apoptosis.

INTRODUCTION

Hepatocellular carcinoma (HCC) is the 6th most frequent cancer in the world and one of the most frequent causes of cancer death.¹⁾ The mortality rate of HCC in Asia along with Africa is very high, especially in underdeveloped areas.²⁾ Among the existing treatment methods, surgical resection or liver transplantation provides the best prognosis, however only 15% of individuals with HCC are appropriate for surgical treatment following initial diagnosis, non-surgical treatment is still needed. Nonetheless, most types of HCC are highly resistant to chemotherapy, and it is often difficult to obtain an ideal therapeutic effect in clinical cases.³⁾ Hence, developing new drugs for treating HCC is a top priority.

The traditional Chinese medicinal plant Sinomenium acutum Rehd. et Wils. (Fam. Menispermaceae) has been used to effectively treat rheumatoid arthritis for centuries.⁴⁾ Sinomenine (SIN) (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methylmorphinan-6-one, C19H23N-O4) is the main effective alkaloid extracted from this plant. It has anti-immune along with anti-inflammatory properties and is extensively utilized in clinically treating rheumatoid diseases.⁵⁾ Investigations have revealed the analgesic mechanisms of sinomenine,^6,7) and its anti-tumor activity is garnering attention. Some research investigations have tested the anti-tumor influence of sinomenine in breast,⁸⁾ liver⁹⁾ and colon cancer.¹⁰⁾ However, the anti-cancer mechanisms of SIN remain unknown.

Our previous investigations have documented that SIN induced glioma cell death via downregulation the protein kinase B (AKT)-mammalian target of rapamycin (mTOR) signaling cascade,¹¹⁾ and Chuan xiongqin Qing tengjian Mixture (CQM) prescription with SIN as the primary active ingredient exhibited a remarkable analgesic influence on cancer pain.¹²⁾ Based on the pharmacological activity of SIN which involves both anti-tumor activities and treating cancer pain, we believe that it has great potential in the clinical treatment of tumors. Based on traditional Chinese medicine (TCM) theory, Sinomenium acutum belongs to the liver meridian. Therefore, this research work examines the effect along with mechanism of SIN against HCC.

The phosphatidylinositol 3-kinase (PI3K)/AKT signaling cascade plays an indispensable role in mediating cell growth and apoptosis.¹³⁾ Research evidence documents that activating the PI3K/AKT signaling cascade enhanced proliferation along with metastasis of HCC cells.^14,15) However, many natural products, such as aloperine and apigenin, can induce apoptosis of HCC cells via downregulating the PI3K/AKT cascade.^16,17) Therefore, modulation of the PI3K/AKT cascade is an effective way to counter the proliferation of HCC. However, whether SIN could regulate the PI3K/AKT pathway against HCC proliferation has not been reported yet.

The present research premise firstly indicated that inhibition of PI3K/AKT1 signaling cascade by SIN could trigger apoptosis of SK-Hep-1 cells, which provides a new scientific basis for the potential new indication of SIN.

MATERIALS AND METHODS

Cell Culture

The human HCC cell SK-Hep-1 was purchased from American Type Culture Collection (ATCC; Manassas, VA, U.S.A.) and inoculated in minimum essential medium (MEM) (GIBCO, NY, U.S.A.) containing 10% fetal bovine serum (FBS) (GIBCO) under 37 °C coupled with 5% CO₂ conditions, until a 75% confluence. After that, we inoculated the cells with specified levels of SIN (Beijing Bethealth People Biomedical Technology Co., Ltd., Beijing, China). Where indicated, insulin-like growth factor-1 (IGF-1) (100 ng/mL) (Selleck Chemicals, Shanghai, China) was added 1 h before SIN administration.

Cell Counting Kit 8 (CCK8) Test

CCK8 (Dojindo, Kumamoto, Japan) was adopted to detect the cell viability. SK-Hep-1 HCC cells were inoculated in 96-well plates and administered with diverse levels of SIN for 24 or 48 h. After treatment, we introduced 10 µL of CCK-8 reagent to every well for one hour. Afterwards, optical density (OD) values were read at 450 nm with the Microplate Reader (Tecan, Männedorf, Switzerland).

Determination of Lactate Dehydrogenase (LDH)

After different concentrations of SIN treated SK-Hep-1 HCC cells for 48 h, as described by the manufacturer, the LDH kit (Roche, Basel, Switzerland) was utilized to measure the LDH release level of damaged cells to evaluate cytotoxicity.

Colony Formation

We adjusted the cell density to 7.5 × 10² cells/mL. We inoculated 2 mL of cell suspension in a six-well plate under 37 °C along with 5% CO₂ conditions. After plating for 24 h, we added diverse levels of SIN for 24 h, discarded the old medium, washed with phosphate buffered saline (PBS), and continued culturing for 10 d to form colonies. Changing of the inoculation medium was done every three days. Finally, fixation of cells colonies was done (in 4% PFA) for 15 min at room temperature (r.t.), followed by staining (in 0.5% crystal violet) for 10 min at r.t..

5-Ethynyl-20-deoxyuridine (EdU) Incorporation Assays

EdU cell proliferation detection kit (Beyotime, Nantong, Jiangsu, China) was used to detect cell division capacity. In short, different concentrations of SIN treated SK-Hep-1 HCC cells for 48 h. The later SK-Hep-1 HCC cells were inoculated with 50 µM EdU for EdU staining per the manufacturer’s instructions. The ration EdU-positive cells to the overall number of Hoechst-positive cells was used as the EdU incorporation rate.

Annexin V/Propidium Iodide (PI) Double Staining

Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit was obtained from BD Company (Franklin Lakes, NJ, U.S.A.) and the Annexin V-FITC/PI staining method was utilized to assess the influence of SIN on SK-Hep-1 HCC cell apoptosis as per the operating instructions. We inoculated the cells in a six-well plate for 24 h followed by inoculation with diverse levels of SIN for 48 h. Collection of the treated cells was done via trypsinization and span at 4 °C and 1000 rpm for five minutes to suspend 1 × 10⁵ cells in 100 µL of 1× binding buffer. Afterwards, we inoculated the cells in the dark with PI (5 µL) along with Annexin V-FITC (5 µL) at r.t. (25 °C) for 15 min. Lastly, we introduced 400 µL of 1× binding buffer, followed by assessment of apoptosis level using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, U.S.A.). The total apoptotic ratio is based on the sum of early apoptosis ratio (Annexin V+/PI−) and late apoptosis ratio (Annexin V+/PI+).

Confocal Microscopy

Terminal deoxynucleotidyl transferase-mediated biotinylated uridine 5′-triphosphate nick end labeling (TUNEL) kit (Roche) was adopted to determine the DNA fragmentation of the SK-Hep-1 HCC cell apoptosis. The cells were cultured on the coverslip for 24 h. After 48 h of SIN treatment, fixation of the cells was done through inoculation with 4% PFA solution for 30 min at r.t. The treated cells were inoculated with 0.3% H₂O₂ in methanol at r.t. for 30 min and afterwards inoculated with 0.1% sodium citrate along with 0.1% Triton X-100 dialysate at 4 °C for three minutes. After that, the cells were inoculated with the TUNEL reaction mixture at 37 °C for one hour and observed with a laser confocal microscope (LSM700, Zeiss, Jena, Germany). We selected five fields at random for viewing of each coverslip to count the TUNEL-positive nuclei and then compared the TUNEL-positive count with the total number of nuclei stained by 4′-6-diamidino-2-phenylindole (DAPI).

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Invitgen, Eugene, U.S.A.) was adopted to assay for mitochondrial membrane potential (MMP). In short, 1 × 10⁵ cells/mL were inoculated completely in a six-well dish and afterwards inoculated in the dark with JC-1 at a final level of 2 mmol/L for 30 min. Thereafter, we rinsed the cells thrice with PBS, and the observation under laser confocal microscope was done (LSM700, Zeiss). The ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers) was used to calculate the MMP of SK-Hep-1 HCC cells.

After sacrificing the mice, the tumor tissues were separated and fixed in 4% PFA for three days. We stained the paraffin sections of the tumor tissues with TUNEL kit (Roche). The apoptotic cell nucleus was stained with green fluorescein and the total number of SK-Hep-1 HCC cell nuclei was marked with DAPI. A laser confocal microscope (LSM700, Zeiss) was used to observe the image of the tumor tissue. The ratio of TUNEL-positive cell nucleus to DAPI-stained cell nucleus was used to express the rate of apoptosis.

Immunohistochemistry

The paraffin sections of tumor tissues were deparaffinized, then rehydration with xylene was done and then using diverse levels of ethanol, and then infiltrated to repair the antigen. Subsequently, we incubated them for 1.5 h with1% bovine serum albumin (BSA) and then incubated them with the primary antibody B-cell leukemia/lymphoma 2 (BCL-2) (1 : 50), AKT1 (1 : 50), PI3K p85α (1 : 50), and proliferating cell nuclear antigen (PCNA) (1 : 50) purchased from Santa Cruz (MA, U.S.A.) (1 : 50) at 4 °C for 16 h. After that, the horseradish peroxidase (HRP)-linked secondary antibody (Abcam, Cambridge, U.K.) was inoculated at 37 °C for one hour. We washed the sections with PBS. We used diaminobenzidine for color reaction and hematoxylin for nuclear staining. The image was taken under a light microscope (Leica DM750, Germany). Brown-yellow coloring indicated positive staining. The expression intensity was measured with ImageJ software.

Western Blotting

The protein extraction kit (Beyotime, Nantong, Jiangsu, China) was utilized to isolate total proteins from tissues and cells. The bicinchoninic acid (BCA) protein concentration detection kit (Biyuntian) was adopted to detect the protein concentration. After loading 25 µg in each loading well, we separated the protein via sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) electrophoresis and transfer-embedded it to polyvinylidene difluoride (PVDF). We inoculated the PVDF with 5% skimmed milk at r.t. for one hour to prevent non-specific binding. After that, we inoculated the membranes overnight with the primary antibodies PI3K p85α (1 : 200, Santa Cruz), AKT1 (1 : 200, Santa Cruz), BCL-2 (1 : 200, Santa Cruz), Caspase 9 (1 : 200, Santa Cruz), Cleaved Caspase 9 (1 : 1000, CST), Caspase 3 (1 : 1000, CST), and Cleaved Caspase 3 (1 : 1000, CST) at 4 °C. The corresponding secondary antibody (1 : 1000, CST) was combined with horseradish peroxide and incubated the membranes for one hour at r.t. We added enhanced chemiluminescence (ECL) and used the Bio-Rad Gel Doc EZ imager (Bio-Rad, CA, U.S.A.) for development. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin (CST, 1 : 1000) was used as the loading control.

Animal Treatments

All animal protocols were carried out as per the procedures approved by the Animal Ethics Committee of the China Academy of Chinese Medical Sciences, Beijing, China. We acquired five-week-old male BALB/c nude mice, weighing about 20 g from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and raised in the specific pathogen-free barrier system of the Animal Center of the Chinese Academy of Chinese Medical Sciences. After one week of acclimatization, SK-Hep-1 HCC cells (1 × 10⁷) were dispersed in 0.2 mL of medium (Matrigel and PBS 1 : 1) and then subcutaneously inoculated into the right side of nude mice. Approximately 24 h after SK-Hep-1 HCC cell transplantation, the mice were stratified at random into three groups, 6 mice per group. The animals of control group were injected intraperitoneally (i.p.) with 5% dimethyl sulfoxide (DMSO) (0.1 mL/10 g body weight) in physiological saline, once daily. SIN was dispersed in physiological saline containing 5% DMSO at a dosage of 75 or 150 mg/kg body weight referring to previous reports,^11,18) once a day, for 20 consecutive days. From the 6th day after SK-Hep-1 HCC cell inoculation, the tumor size was assessed by a caliper every three to four days, we computed the tumor volume as per the standard formula: (width² × length)/2 and expressed as mm³. We also monitored weight changes in the nude mice. Lastly, we sacrificed the mice on the 20th day after SK-Hep-1 HCC cell inoculation. We then obtained tumor tissues, weighed and photographed them, and snap-frozen them in liquid nitrogen for Western blotting or fixed them (in 4% PFA) for immunohistochemical assessment.

Evaluation of Biochemical Parameter

At the end of the in vivo experiments, the whole blood was taken from the mouse orbit, and placed at r.t. for 45 min, then span at 3500 rpm at r.t. for 10 min. Thereafter, the serum was separated and the contents of glutamic-pyruvic transaminase (ALT) along with glutamic oxaloacetic transaminase (AST) were determined as documented in the manufacturer kits (Nanjing Jiancheng Bioengineering Institute, China).

Statistical Analysis

All data analyses were implemented in the GraphPad Prism 8.0.1 software (San Diego, CA, U.S.A.) and the data are given as mean ± standard deviation (S.D.). The comparison among the groups was calculated by one-way ANOVA (i.e., Sidak multiple comparisons test), with p=<0.05 signifying statistical significance.

RESULTS

SIN Inhibits the Viability of Hepatocellular Carcinoma Cells

To detect the effect of SIN (Fig. 1A) on HCC cell viability, we treated SK-Hep-1 HCC cells with diverse levels of SIN (0.0625, 0.125, 0.25, 0.5, and 1 mM) for 24 or 48 h, using PBS containing 1/1000 DMSO as a control. CCK8 data illustrated that SIN reduced the survival rate of SK-Hep-1 HCC cells in a dose-, as well as time-based approach (p < 0.01) (Fig. 1B). LDH is a living cell cytoplasmic enzyme, which cannot release cytoplasm under normal conditions. The amount of LDH released in the medium is directly proportional to the number of injured cells. The LDH assays indicated that SIN (0.125, 0.25, 0.5, and 1 mM) treatment for 48 h increased the damage of SK-Hep-1 in a dose-dependent way (p < 0.01) (Fig. 1C). To further assess the influence of SIN on the replication ability of HCC cells, we used the clone formation test to detect the replication activities. The test revealed that after SIN (0.125, 0.25, and 0.5 mM) treatment, the number of colonies was remarkably reduced in contrast with the controls (Fig. 1D), illustrating that SIN reduced the replication ability of HCC cells. Besides, we observed the effect of SIN on the division capacity of HCC cells through EdU incorporation experiments. EdU labeling showed that in contrast with the controls, that SIN (0.125, 0.25, and 0.5 mM) treated SK-Hep-1 HCC cells for 48 h dose-dependently attenuated the division of HCC cells (p < 0.01) (Fig. 1E). The above observations illustrated that SIN could effectively reduce the proliferation of SK-Hep-1 HCC cells.

Fig. 1. SIN Suppresses the Proliferative Activity of SK-Hep-1 Cells

(A) The chemical structure of SIN. (B) CCK8 experiments exhibited that SIN dose- along with time-dependently reduced the cell survival rate of SK-Hep-1 cells. Following the inoculation of SK-Hep-1 cells with diverse levels of SIN (0.0625, 0.125, 0.25, 0.5, and 1 mM) for 24 and 48 h, we assessed cell viability via the CCK8 assays. (C) LDH experiments showed that SIN could dose-dependently increase cytotoxicity of SK-Hep-1 cells. After SK-Hep-1 cells were inoculated with 0.125, 0.25, 0.5 and 1 mM SIN for 48 h, we detected the amount of LDH released. (D) Colony formation experiments showed that SIN reduced the replication capacity of SK-Hep-1 cells, dose-dependently. SK-Hep-1 cells were inoculated with SIN (0.125, 0.25, or 0.5 mM) for 24 h and the cell replication ability was tested by colony formation experiments. (E) EdU incorporation experiments showed that SIN dose-dependently reduced the cell division capacity of SK-Hep-1 cells. Following 48 h of treatment with SIN (0.125, 0.25, or 0.5 mM), EdU incorporation experiments were performed to detect cell division capacity. All data are given as mean ± S.D., n = 3. (**) p < 0.01 in contrast with the controls.

SIN Triggered Apoptosis of HCC Cells

Apoptosis is the main mechanism by which anti-tumor drugs work. To explore why SIN inhibits the proliferation of HCC cells, this study used staining with annexin V/PI and TUNEL staining to assess apoptosis. The results of annexin V/PI double staining depicted that after 48 h of SIN treatment (0.125, 0.25, and 0.5 mM), the ratio of the annexin V positive cells in each administration group was remarkably higher in contrast with that of the controls in a dose-based approach (p < 0.01) (Figs. 2A, B). TUNEL staining results revealed that after SK-Hep-1 HCC cells were treated by SIN (0.125, 0.25, and 0.5 mM) for 48 h, the ratio of TUNEL positive cells in each administration group was remarkably higher in contrast with that of the controls in a dose-based approach (p < 0.01) (Figs. 2C, D). Besides, as exhibited in Fig. 3A, detection of the apoptosis marker protein Cleaved Caspase 3 via Western blotting suggested that after SIN (0.125, 0.25, and 0.5 mM) treated SK-Hep-1 HCC cells for 48 h, the expression level of Pro-Caspase 3 was remarkably reduced and Cleaved Caspase 3 expression was drastically elevated in contrast with the controls (p < 0.01). The above results demonstrated that SIN triggered apoptosis in HCC cells, thereby decreasing the proliferation of SK-Hep-1 HCC cells.

Fig. 2. SIN Induced SK-Hep-1 Cell Apoptosis

(A) Double staining (in annexin V/PI) depicted that SIN increased the rate of apoptosis of SK-Hep-1 in a dose-based approach. After treating SK-Hep-1 cells for 48 h with diverse levels of SIN (0.125, 0.25, or 0.5 mM), we assayed the apoptosis level via flow cytometry and then statistically analyzed (B). (C) The representative TUNEL staining images showed that SIN up-regulated the apoptosis level of SK-Hep-1 cells, dose-dependently. After SK-Hep-1 cells were inoculated with SIN (0.125, 0.25, or 0.5 mM) for 48 h, the apoptosis level was detected by TUNEL staining and then statistically analyzed (D). All data are given as mean ± S.D., n = 3. (**) p < 0.01 vs. controls.

Fig. 3. SIN Promotes SK-Hep-1 Cell Apoptosis by Triggering Mitochondrial Stress

(A) Western blot illustrated that SIN (0.125, 0.25, or 0.5 mM) treating SK-Hep-1 cells for 48 h dose-dependently reduced the contents of BCL-2 along with Pro-Caspase 9/3, and increased Cleaved Caspase 9/3 expressions, with β-actin serving as the loading standard. (B) The representative JC-1 staining images showed that SIN induced the MMP collapse of SK-Hep-1 cells, dose-dependently. After 24 h of treatment with SIN (0.25 or 0.5 mM), the level of MMP in SK Hep-1 cells was assessed via staining (in JC-1). All data are given as mean ± S.D., n = 3. (**) p < 0.01 in contrast with the controls.

SIN Activates the Mitochondrial-Mediated Apoptosis Cascade in HCC Cells

The molecular regulation of mechanisms involved in apoptosis are very complicated. To elucidate the upstream mechanism of SIN-triggered apoptosis of HCC cells, we conducted the following experiments. Western blotting demonstrated that after 48 h of SIN treatment (0.125, 0.25, and 0.5 mM), the expression levels of BCL-2 closely related to the mitochondrial-mediated apoptosis pathway and Pro-Caspase 9/3 were considerably reduced, and Cleaved Caspase 9/3 expressions were strongly elevated, in contrast with the controls. (p < 0.01) (Fig. 3A).

To confirm that SIN-induced apoptosis of HCC cells is mediated by mitochondrial, we performed JC-1 staining to observe the level of mitochondrial stress in each experimental group. JC-1 is an ideal fluorescent probe extensively employed to explore the mitochondrial membrane potential (MMP) ∆Ψm. If the MMP is high, JC-1 gathers in the matrix of the mitochondria to generate aggregates, which can give red fluorescence. If the MMP is low, JC-1 cannot accumulate in the matrix of the mitochondria, it becomes monomers that produces green fluorescence. Hence, the change in MMP could be detected by the shift in fluorescence color. The experimental results exhibited that after 24 h of inoculation with diverse levels of SIN (0.25 and 0.5 mM), in contrast with the controls, the JC-1 gathered in the mitochondria changed from aggregates to monomers form, there was a fluorescent transition from red to green, and the ratio of red fluorescence to green fluorescence reduced dramatically (p < 0.01) (Fig. 3B), indicating that the MMP decreased and depolarization elevated. The above results illustrated that SIN induced the activation of endogenous apoptosis pathways mediated by mitochondria in HCC cells.

PI3K/AKT1 Signaling Cascade Mediates SIN-Induced Mitochondria-Triggered Endogenous Apoptosis of HCC Cells

The PI3K/AKT cascade is a pivotal modulator of cell apoptosis, according this, we examined whether this pathway participates in apoptosis of SK-Hep-1 HCC cells triggered by SIN. Using Western blotting, we found that after 48 h of SK-Hep-1 HCC cells being inoculated with SIN (0.125, 0.25, or 0.5 mM), the PI3K p85α, and AKT1 contents were remarkably decreased in contrast with the controls (p < 0.01) (Fig. 4A). More importantly, after pretreatment with IGF-1 for 1 h and co-inoculation with SIN for 48 h, Western blotting data exhibited that IGF-1 (100 ng/mL) evidently reversed the decrease in p-AKT1 ^(Ser473) expression induced by SIN (0.5 mM) treatment alone in SK-Hep-1 cells as well as the decrease in BCL-2 expression, which is a pivotal biomolecule in the mitochondrial-mediated apoptosis cascade (p < 0.01) (Fig. 4B). Besides, the results of annexin V/PI double staining revealed that IGF-1 (100 ng/mL) pretreatment markedly reversed the high ratio of apoptosis triggered by SIN treatment alone (p < 0.01) (Figs. 4C, D). The above findings illustrated that inhibition of PI3K/AKT1 pathway was involved in SIN-triggered mitochondria-mediated apoptosis of SK-Hep-1 cells.

Fig. 4. Inhibition of PI3K/AKT1 Signaling Cascade by SIN Induces Mitochondrial-Mediated Apoptosis of SK-Hep-1 Cells

(A) Western blot exhibited that inoculation of SK-Hep-1 cells with SIN (0.125, 0.25, or 0.5 mM) for 48 h in reduced the contents of PI3K p85α, and AKT 1 in a dose-dependent approach. β-Actin was employed as the loading standard. (B) Western blot illustrated that after IGF-1 (100 ng/mL) pretreated SK-Hep-1 cells for one hour and co-treated SK-Hep-1 cells with SIN (0.5 mM) for 48 h, the decrease in p-AKT1 ^(Ser473) and BCL-2 expression induced by SIN (0.5 mM) was remarkably reversed. GAPDH was utilized as the loading standard. (C) Annexin V/PI double staining results illustrated that pre-treatment of SK-Hep-1 cells for one hour with IGF-1 (100 ng/mL) and co-treatment using SIN (0.5 mM) for 48 h remarkably reversed the increase in apoptotic rate of SK-Hep-1 cells induced by SIN (0.5 mM) treatment alone, and the result from statistical analyses was described by column charts (D). All data are given as the mean ± S.D., n = 3. (**) p < 0.01 vs., (&&) p < 0.01 vs. the SIN group.

SIN Suppresses Tumor Growth in Vivo

To clarify the pharmacodynamic effect of SIN against HCC in vivo, we subcutaneously inoculated SK-Hep-1 HCC cells in nude mice and provided 150 and 75 mg/kg of SIN once per day. After continuous administration for 20 d, we assessed the influence of SIN on the growth of mouse xenograft tumors. The experimental results illustrated that in contrast with the controls, SIN could reduce tumor weight (Figs. 5A, B) and volume (Fig. 5C) in a dose-based approach (p < 0.01), and had no impact on the bodyweight of nude mice (Fig. 5D). Immunohistochemical suggested that SIN (75 and 150 mg/kg) dose-dependently reduced the expression of the PCNA in tumor tissues, which characterizes the level of cell proliferation (p < 0.01) (Fig. 5E). The above data illustrated that SIN could suppress the growth of HCC tissue in vivo. Moreover, there was no remarkable difference in the content of ALT (Fig. 5F) along with AST (Fig. 5G) among the different groups, and we found no abnormal behavior during the treatment, as well as no morphological abnormalities in major organs for instance the liver, lung, spleen, and kidney.

Fig. 5. SIN Inhibits the Proliferation of Tumor in Vivo

We inoculated SK-Hep-1 cells subcutaneously in nude mice, and SIN (150 or 75 mg/kg) was continuously administered the next day, once a day. From the sixth day after inoculation, the tumor volume was measured once every three to four days. We sacrificed the mice 20 d later after inoculation, and the tumor tissues were separated and photographed (A). (B) The tumor tissue weight was weighed and recorded. (C) The tumor volume change was measured. (D) The nude mouse body weight change was also counted. (E) The results of representative immunohistochemistry images illustrated that SIN (75 and 150 mg/kg) in a dose-dependent approach diminished the expression of PCNA in tumor tissues. Contents of ALT (F) and AST (G) of mice were observed. All data are given as mean ± S.D., n = 6. (**) p < 0.01 in contrast with the controls; NS, no significance versus the control group.

SIN Triggered the Mitochondrial Apoptosis Pathway Mediated via PI3K/AKT1 Signaling in HCC in Vivo

To verify the in vitro experiments’ conclusions, we performed TUNEL staining and found that, in contrast with the controls, SIN (75 and 150 mg/kg) dose-dependently increased the apoptosis level of HCC tumor tissues (p < 0.01) (Fig. 6A). This result illustrated that SIN triggered the apoptosis of HCC cells in vivo. We then used immunohistochemistry and immunoblotting to explore the expression levels of the key signal molecules in PI3K/AKT1 signaling-mediated mitochondrial apoptosis pathway. The results of the immunohistochemistry showed that inn contrast with the controls, SIN (75 and 150 mg/kg) dose-dependently diminished the contents of PI3K p85α, AKT1 and BCL-2 in HCC tumor tissues (p < 0.01) (Fig. 6B). Moreover, Western blot analysis illustrated that SIN (75 and 150 mg/kg) dose-dependently down-regulated the expression levels of PI3K p85α, AKT1, BCL-2, Pro-Caspase-9, along with Pro-Caspase-3 in HCC tumor tissues and up-regulated Cleaved Caspase-9, as well as Cleaved Caspase-3 expressions (p < 0.01) (Fig. 6C). These results were in line with in vitro experimental results.

Fig. 6. SIN Regulates PI3K/AKT1 Signaling-Mediated Endogenous Apoptosis Triggered by Mitochondria of HCC in Vivo

Tumors were dissected from mice inoculated with vehicle or SIN (150 or 75 mg/kg) as shown in Fig. 5A. (A) Representative images of TUNEL staining exhibited that SIN (75 and 150 mg/kg) escalated the apoptosis levels of tumor tissues in a dose-based approach. (B) The results of representative immunohistochemistry images exhibited that SIN (75 and 150 mg/kg) dose-dependently diminished the contents of PI3K p85α, AKT1 and BCL-2 in tumor tissues. (C) Western blot analysis illustrated that SIN (150 and 75 mg/kg) dose-dependently reduced the contents of PI3K p85α, AKT1, BCL-2, as well as Pro-Caspase 9/3, and increased the expression content of Cleaved Caspase 9/3. β-Actin served as the loading standard. All data are given as mean ± S.D., n = 3. (**) p < 0.01 in contrast with the controls.

In summary, SIN interfered with the same signaling pathway in vivo as in vitro. That is, SIN promoted mitochondria-mediated apoptosis in HCC via downregulating the PI3K/AKT1 signaling cascade.

DISCUSSION

Chinese herbal medicine has attracted attention for the development of natural anti-cancer drugs. A previous investigation documented that SIN prevented the proliferation of glioma cells through the intervention in the AKT signaling,¹¹⁾ and was less toxic to normal cells.¹⁹⁾ In this study, based on TCM theory, we examined the effect and mechanism of SIN against HCC, and firstly found that PI3K/AKT1 signaling cascade mediates SIN-triggered apoptosis of SK-Hep-1 cells.

Uncontrolled cell proliferation is an important sign of tumorigenesis and development. Hence, suppressing tumor cell growth or enhancing tumor cells apoptosis known as “type 1 programmed cell death” is an important goal in averting tumor progress.²⁰⁾ Herein, the effect of SIN on SK-Hep-1 cell apoptosis was explored. We used CCK8, LDH, colony formation and EdU incorporation experiments to confirm that SIN can reduce the cell survival rate, damage degree, replication ability and division level of SK-Hep-1. In vivo assays exhibited that SIN reduced the tumor volume along with tumor weight of tumor-bearing nude mice, and decreased the expression level of PCNA in tumor tissues, but did not remarkably affect body weight of mice. More importantly, TUNEL staining and double staining with annexin V/PI revealed that SIN induced SK-Hep-1 cell apoptosis in a dose-dependent approach, which was congruent with the results of TUNEL staining in tumor tissues. Therefore, SIN induced SK-Hep-1 cell apoptosis, which was consistent with previous study.²¹⁾

The BCL-2, and Caspase family are distinct modulatory proteins of the mitochondria-mediated apoptosis cascade that serve as key regulators of apoptosis.²²⁾ Therefore, we speculated that the mitochondria apoptosis cascade participates in the SIN-triggered apoptosis of SK-Hep-1 cells. The mitochondria-dependent endogenous apoptosis cascade is mediated via intracellular signals converging at the mitochondrial level to respond to diverse stress factors (such as chemotherapy drugs, etc.).²³⁾ Subsequently, the antiapoptotic protein BCL-2 is neutralized, which disrupts permeability of the mitochondrial membrane. This allows proteins normally located in the interstitial space to diffuse into the cytoplasm, thereby promoting the formation of apoptosomes which recruits the trigger precursor Pro-Caspase-9 to its caspase recruit domains to perform self-activation. This in turn activates the downstream executors Caspases-3 to lyse intracellular substrates, leading to mitochondria-mediated apoptotic cell death.^24,25) Herein, Western blot data showed that SIN dose-dependently reduced the contents of BCL-2, and increased the contents of Cleaved Caspase 9/3 in vitro along with in vivo. The results of immunohistochemistry illustrated that SIN remarkably reduced the content of BCL-2 in tumor tissues. To further confirm that SIN induced SK-Hep-1 cell apoptosis via the mitochondrial apoptotic pathway, we performed JC-1 staining test. The data illustrated that in contrast with the controls, as the dose of SIN increased, the intensity of fluorescence of JC-1 monomers increased strongly, whereas the intensity of fluorescence of JC-1 aggregates decreased obviously, indicating that SIN induced collapse of MMP of SK-Hep-1 cells in a dose-based way. Combined with the above, we concluded that SIN triggered the stimulation of the mitochondrial apoptosis cascade in SK-Hep-1 cells. Additional literature reported that SIN activated the mitochondrial apoptosis pathway in Hep3B and SMMC7721 cells, which was congruent with the findings of this study.⁹⁾

The PI3K/AKT cascade is a pivotal intracellular mediator pathway crucial for the modulation of cell proliferation along with apoptosis.^26,27) PI3K constitutes an intracellular phosphatidylinositol kinase that plays an indispensable role in numerous cell functions, for instance differentiation, cell proliferation, as well as apoptosis.²⁸⁾ AKT is a pivotal downstream effector of PI3K, which plays key roles through the phosphorylation of numerous downstream signaling cascades, e.g., the BCL-2 family and caspase-dependent cascades.²⁹⁾ More specifically, AKT is also called protein kinase B (PKB), in which three PKB family members have been isolated, referred as PKBα (AKT1), PKBγ (AKT3), as well as PKBβ (AKT2).³⁰⁾ Each isomer has a pleckstrin homology domain composed of 100 amino acids at the N-terminus. Then there is a kinase domain, which remarkably resembles protein kinase A (PKA) and PKC.^31,32) There is a hydrophobic tail at the C-terminal behind the kinase domain that contains a regulatory phosphorylation site (Ser473 in AKTl). Phosphorylation of Ser473 is a response to growth factors, as well as other extracellular stimuli, which is necessary to maximize AKT activation.³³⁾ Our previous investigations have documented that SIN induced glioma cell death via AKT signaling.¹¹⁾ Based on the above studies, we speculated that PI3K/AKT1 signal pathway may be involved in SIN-induced apoptosis of SK-Hep-1 cells. Therefore, we analyzed the possible signal transduction cascades during SIN-mediated apoptosis of SK-Hep-1 cells via Western blotting. Interestingly, we revealed that SIN diminished the contents of ATK1 and PI3K p85α in HCC cells, in vitro along with in vivo in a dose-dependent approach. Immunohistochemistry analysis also showed that the contents of ATK1 and PI3K p85α in tumor tissues were remarkably diminished after SIN treatment. Even though the reasons for the down-regulation of PI3K p85α and AKT1 expression induced by SIN maybe complex, they could eventually promote the down-regulation of p-AKT1^(Ser473) expression, resulting in a downstream cascade reaction. Hence, SK-Hep-1 cells were pretreated with IGF-1 to determine whether PI3K/AKT1 signaling pathway was involved in SK-Hep-1 cell apoptosis induced by SIN. IGF-1, as we all known, is a powerful activator of the PI3K/AKT pathway, binding of IGF-1 to the IGF-1 receptor results in phosphorylation of PI3K, which, in turn, phosphorylates AKT.^34–36) The results showed that when SK-Hep-1 cells were stimulated by IGF-1 and SIN at the same time, the contents of p-AKT1^(Ser473) along with BCL-2 were increased obviously and the apoptosis rate was strongly lower than that of the SIN treatment alone. It meant that HCC cells’ apoptosis induced by SIN was mediated via downregulating the PI3K/AKT1 signaling cascade, which was similar to other tumor cells investigated in previous research works, such as prostate cancer,³⁷⁾ lung cancer³⁸⁾ and retinoblastoma.³⁹⁾ What is exciting is that our study has more clearly confirmed the regulatory effect of SIN on AKT1 of the PI3K/AKT signal pathway. All the above results strongly proved that AKT1 could work as a target of anticancer drugs. Considering the effect of SIN on AKT1, except for anti-tumor effect, the other biological activity of SIN in different animal models of experimental diseases remains to be further studied. Additionally, our previous study has shown that SIN induced autophagic death of glioma cells, rather than apoptosis, by the AKT signaling.¹¹⁾ It is suggested that the effects of SIN on different tumor cell lines are different. More importantly, whether SIN could trigger autophagy of SK-Hep-1 cells, whether the inhibition of PI3K/AKT pathway is involved in it and whether this kind of autophagy is protective to cells or related to the promotion of cell death deserve to be explored in depth later.

In summary, we proved that SIN, as a key inhibitor of HCC growth, is related to the stimulation of cell apoptosis. More research on the mechanisms have shown that the activation of the mitochondrial apoptosis cascade and the repression of the PI3K/AKT1 cascade may be the primary mechanisms involved in anti-HCC effect of SIN. Most importantly, our findings provided a new molecular mechanism for the treatment of HCC by SIN (Fig. 7). Meanwhile, we supplied a novel theoretical basis for its prospective clinical utility in treating HCC. Next research should focus on using more HCC cell lines and add positive drugs (Sorafenib) and AKT1 agonists in in vivo experiments to further corroborate our findings.

Fig. 7. Schematic Diagram Illustrating the Mechanism of SIN against the Proliferation of HCC Cells

Acknowledgments

This study was supported by China Postdoctoral Science Foundation funded project (No. 2020M680834), Jiangxi Provincial Natural Science Foundation (No. 20212BAB216002), and The Research Fund of Gannan Medical University (No. YB201911; No. QD201821).

Author Contributions

Conceived and designed the experiments: Yumao Jiang, Jihua Zhao.

Performed the experiments: Yan Luo, Liwei Liu.

Analyzed the data: Yue Jiao, Guangli Xu, Meiyu Zhang.

Contributed reagents/materials/analysis tools: Yumao Jiang, Jihua Zhao.

Wrote and revised the paper: Yumao Jiang, Yan Luo.

Conflict of Interest

The authors declare no conflict of interest.

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