Cryptotanshinone Induces Apoptosis and Inhibits Migration and Invasion in Human Hepatocellular Carcinoma Cells In Vitro

Abstract

Cryptotanshinone (CPT), an active quinoid diterpene isolated from Salvia miltiorrhiza Bunge, was previously reported to have potential anticancer effects. However, the mechanisms of CPT on hepatocellular carcinoma (HCC) cells are not well understood. In this study, we investigated the anticancer effects of CPT on HCC cells. Thiazolyl blue tetrazolium bromide assay showed dose-dependent and time-dependent cytotoxicity of CPT on human HCC cells, especially in HCCLM3 and Huh-7 cells. Hoechst 33258 stain, flow cytometry assay, and Western blot assay all indicated that CPT could distinctly induce the apoptosis of human HCC cells and break intracellular homeostasis by triggering the imbalance of mitochondrial transmembrane potential (Δψm) and reactive oxygen species. In addition, CPT could significantly inhibit HCCLM3 and Huh-7 cells’ migration and invasion via the signal transducers and activators of transcription 3/matrix metalloproteinases mediated signaling pathway. Our findings demonstrated that the antitumor effects of CPT on human HCC cells were by suppressing cell proliferation, inducing cell apoptosis and impairing cell migration and invasion.

Hepatocellular carcinoma (HCC) is a kind of pernicious liver tumor that accounts for 75% to 85% of primary liver cancers. ¹ HCC, which is the sixth most prevalent cancer and the fourth leading cause of cancer deaths worldwide, has a very poor prognosis with a 5-year survival rate of less than 5% in most symptomatic patients. Based on global cancer statistics, 841 000 new liver cancer cases and 782 000 deaths occurred worldwide during 2018, and China is one of the most high-risk HCC areas. ¹ Although significant progress has been made in HCC, high-metastatic potential and proverbially high resistance to conventional therapies have resulted in poor prognosis. Therefore, it is urgent to develop novel therapies with high reliability and safety.

There is growing evidence that ingredients in the human diet can reduce the risk of cancer. ² DanShen (Salvia miltiorrhiza Bunge, family Lamiaceae) is a traditional Chinese medicinal herb with a history of nearly 2000 years. Cryptotanshinone (CPT, Figure 1(a)), a diterpenoid quinone isolated from Danshen, has antioxidant, anti-inflammatory, and antibacterial effects. CPT has been reported to inhibit many tumors in recent years, such as tongue squamous cell carcinoma, ovarian cancer, lung tumors, colorectal cancer, and renal cell carcinoma. ^{3 -7} Great progress has been made in the study of the pharmacological mechanism of CPT.

Figure 1

Structure of cryptotanshinone (CPT) and cytotoxic effects on multiple hepatocellular carcinoma (HCC) cell lines. (a) Structure of CPT; (b) SMMC-7721; (c) PLC/PRF/5; (d) SK-hep-1; (e) QGY-7701; (f) HepG-2; (g) Huh-6; (h) HCCLM3; (i) Huh-7; (j)LO2; (k) Colony formations in HCCLM3 and Huh-7 cells; and (l) the statistic results of colony formation assays; *P < 0.05 vs control group; **P < 0.01 vs control group; ***P < 0.001 vs control group.

Specifically, in many malignant tumors, signal transducers and activators of transcription 3 (Stat3) are considered to play a carcinogenic role, participating in the occurrence and development of human cancers via inducing Stat3 downstream genes that encode apoptosis-related proteins. ⁸ As a Stat3 inhibitor, CPT has been shown to have the ability to inhibit the phosphorylation activity of Stat3. ⁹ Some researchers found that CPT could inhibit the activation of the Stat3 pathways in colorectal cancer cells. ⁷ Moreover, CPT was reported to enhance the efficacy of arsenic trioxide in meliorating liver cancer both in vitro and in vivo via downregulation of phosphorylated-Stat3 expression. ¹⁰ What’s more, as an effective stimulator of endoplasmic reticulum (ER) stress, CPT might lead to apoptosis in various cancer cell lines, including HepG2. ¹¹ However, the precise antitumor mechanism of CPT on some specific HCC cells has not been elucidated.

Considering the broad-spectrum of antitumor effects, we hypothesized that CPT might be practicable for inhibiting the growth of HCC cells. To verify this hypothesis, we treated HCC cells with CPT and studied the related mechanisms. Our results showed that CPT could inhibit the growth of HCCLM3, Huh-7, and another 6 kinds of HCC cell lines. CPT also induced apoptosis and suppressed migration and invasion in HCCLM3 and Huh-7 cells. Considering its safety and effectiveness, these data suggested that CPT might be an agent for the treatment of HCC.

Materials and Methods

Chemicals

CPT was purchased from Chengdu Synguider Technology Co., Ltd. (Chengdu, China); Lot number: SYW12064; and thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), rhodamine-123 (Rh123), and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) from Sigma (St Louis, MO, USA). The primary antibodies used in this research were: β-actin (ZSGB-BIO, Beijing, China), Bax, Bcl-2, cleaved-caspase-3 (Cell Signaling Technology Company, MA, USA), TIMP-2, MMP-9, MMP-2 (Millipore, CA, USA), Stat3, and p-Stat3 (Cell Signaling Technology Company, MA, USA). The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit and Hoechst 33 258 were purchased from KeyGen Biotech (Nanjing, China).

Cell Culture

Eight kinds of HCC cell lines and hepatocyte line LO2 were researched. HepG2 (human hepatoblastoma cells, obtained from ATCC), HCCLM3 (highly metastatic human HCC cells, obtained from CCTCC), Huh-6 (human hepatoblastoma cells, obtained from RIKEN), Huh-7 (human hepatoblastoma cells, obtained from RIKEN), PLC/PRF/5 (human HCC cells (Alexander), obtained from ATCC), and LO2 were cultured in DMEM/high glucose mixed with 10% fetal bovine serum (FBS; Hohhot Cao Yuan Lv Ye Bio-engineering Materials Co. Ltd.) and 1% antibiotics (penicillin and streptomycin, MP Biomedical LLC). The SK-hep-1 (human hepatic ascites adenocarcinoma cells, obtained from ATCC), SMMC-7721 (human hepatoma cells, obtained from CCTCC), and QGY-7701 (human hepatoma cells, obtained from CCTCC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Hohhot Cao Yuan Lv Ye Bio-engineering Materials Co. Ltd.) and 1% antibiotics (penicillin and streptomycin, MP Biomedical LLC). All cell lines were incubated at 37°C in a humidified atmosphere with 5% carbondioxide.

Cell Viability and Colony Formation Assay

Cell viability was evaluated using the MTT assay in compliance with the manufacturer’s instructions. A total of 2000-4000 cells/well was seeded in 96-well plates, with 100 µL/well, and treated with gradient concentrations of CPT after 24 hours. After incubation for 24, 48, and 72 hours, respectively, 20 µL of MTT solution (5 mg/mL) was added to each well and left for an additional 2-4 hours in the incubator. The supernatant was subsequently removed thoroughly before 150 µL of DMSO was used to dissolve the purple-colored formazan precipitates. The absorbance was measured at 570 nm with a microplate spectrophotometer (Molecular Devices, CA, USA).

Cells were seeded into 6-well plates (500 cells/well) and treated with gradient concentrations of CPT after 24 hours incubation. The medium containing CPT was replaced every 3-4 days. About 10 days later, the supernatant was removed, and cells were washed with phosphate-buffered saline (PBS) followed by fixing with methanol for 10 minutes and staining with a 0.5% crystal violet solution for 20 minutes. The colonies were counted under a microscope.

Wound-Healing Migration Assay

In order to assess the ability of cell migration, a wound-healing assay was conducted, as described previously. ¹² When the cancer cells in a 6-well plate had grown to 80% confluence, cell monolayers in the central region were scraped off using sterile 200 µL pipette tips, and the culture was washed with PBS 3 times to remove the delimited cells and then added to 5% serum medium with different concentrations of CPT. After 48 hours incubation, cells were photographed under a microscope (Olympus, IX73, Japan); the percentage inhibition of migrated cells was expressed using 100% as the value of the control group.

Morphological Analysis by Hoechst 33258 Staining

Apoptosis is a familiar form of programmed cell death. An apoptotic cell exhibits special morphological characteristics such as cell contraction, chromatin condensation, margination and the formation of apoptotic bodies, which contain fragments of the nucleus. ¹³ To determine whether the decrease in cell viabilities induced by CPT is attributable to apoptosis, we stained HCC cells with Hoechst 33258 fluorescent dye. ¹⁴ For morphological analysis of the nucleus, HCC cells (1 × 10⁵ cells/wall) were grown on 18 × 18 mm glass coverslips in a 6-well culture-plate overnight. After treatment with a series density of CPT for the following 48 hours, cells were washed with cold PBS 3 times and fixed with methanol for 15 minutes at room temperature, followed by staining with Hoechst 33 258 solutions for another 15 minutes. The nuclear morphological changes were analyzed using fluorescence microscopy (Olympus, BX53, Japan).

Cell Apoptosis Analysis by Flow Cytometry

The effect of CPT on the apoptosis rate of Huh-7 and HCCLM3 cells was tested using an Annexin V-FITC/PI kit. Briefly, cells in a quantity of 1 × 10⁵ cells/well were treated with various CPT concentrations (0-20 μM) for 48 hours. After digestion and harvesting, cells were washed with cold PBS twice. Cell suspensions were cultured with 5 µL Annexin V-FITC at 4°C for 30 minutes, protected from light. Then, 5 µL of PI was added to each well and incubated in the dark for 5 minutes at 37°C. Cells were detected by flow cytometry (FCM); the data were analyzed with FlowJo software.

Mitochondrial Membrane Potential (Δψ_m ) Assay and Reactive Oxygen Species

Mitochondrial membrane potential was measured by FCM using Rh123 staining. ¹⁵ After treating with various concentrations of CPT for 48 hours, cells were digested, collected, washed with cold PBS twice, and dyed with 200 µL Rh123 (10μM) at 37°C for 30 minutes, shielded from light. The cells were then suspended in 1 mL of PBS and analyzed immediately using a flow cytometer with a 488 nm laser. For intracellular reactive oxygen species (ROS) detection, fluorescent probe DCFH-DA staining was used. Cells were treated with different concentrations of CPT for 48 hours, then collected and mixed with 400 µL DCFH-DA (10 µM) diluted in PBS at 37°C in the dark for 30 minutes. ¹⁶ Cells were suspended in 1 mL of PBS and analyzed using a flow cytometer with a 488 nm laser; the data were analyzed with FlowJo software.

Boyden Chamber Migration and Invasion Assay

Migration and invasion abilities of HCC cells were analyzed by the Boyden chamber assay, according to a previously described method. ¹² For migration, cells were trypsinized and seeded in the upper chamber at a density of 1 × 10⁵ cells in 100 µL serum-free medium, while 600 µL medium containing 10% FBS was added at the bottom. Various concentrations of CPT were added to both chambers for 48 hours. The matrix glue and the cells in the upper chamber were gently wiped off with a cotton swab. The chambers were thoroughly air-dried at room temperature to facilitate 0.5% crystal violet staining. The migrated cells were counted and photographed using a light microscope.

For the invasion assay, Matrigel (BD Biosciences) was diluted with a serum-free medium at a ratio of 1:6. After dilution, 60 µL/well was added into the upper chamber of the transwell, while the bottom of the chambers was filled with 600 µL medium containing 10% FBS. After being left at 37°C for 30 minutes until Matrigel polymerization, 100 µL serum-free medium suspension containing 1 × 10⁴ cells and various concentrations of CPT were seeded into the upper chamber. After incubating for 48 hours, the matrix glue and the non-migrated cells in the upper chamber were gently wiped off with a cotton swab, and the chambers thoroughly air-dried at room temperature to facilitate staining with 0.5% crystal violet. The transwell chamber was observed and photographed under a light microscope and cell counts were performed in randomly selected visual fields.

Western Blot Analysis

Western blot analysis was conducted as described previously. ¹⁷ The first-phase preparation was protein sample pretreatment. HCCLM3 and Huh-7 cells were treated with CPT in gradient concentrations for 48 hours before the cells were gathered and washed twice with cold PBS, lysed in radioimmunoprecipitation assay buffer followed by ultrasonic cytolysis and centrifugation. Protein expression concentrations were tested using a bicinchoninic acid Protein Assay Kit (Beyotime Institute of Biotechnology) and quantified before loading. Equal amounts of protein sample were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, followed by transferring onto polyvinylidene difluoride membranes (Amersham Bioscience, NJ, USA). Then, the specific membranes were blocked with 5% non-fat milk at 37°C for 2 hours and incubated with the corresponding primary antibodies overnight at 4°C. After washing the membranes with Tris-buffered saline/Tween 20 (0.1%) 5 times, they were incubated with the appropriate secondary antibodies; the reactive bands appeared using an enhanced chemiluminescence kit (Amersham).

Statistical Analysis

GraphPad Prism 5.0 was used to analyze the data. Data are represented as the average standard deviation (SD) of three independent experiments. The two-tail t-test was used for statistical analysis, and P values were labeled as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Results

CPT Inhibited HCC Cell Proliferation

MTT assay was used to determine the anticancer activities of CPT (Figure 1(a)) by treating 8 HCC cell lines with different concentrations (0, 1.25, 2.5, 5, 10, and 20 µM) for 24, 48, and 72 hours, as displayed in Figure (b-i). Hepatocyte line LO2 was used to observe cytotoxicity (Figure 1(j)). The half-maximal inhibitory concentration value of CPT for each cell line is shown in Table 1. CPT induced growth inhibition in time-dependent and dose-dependent manners and the form of inhibition was cell-type dependent. Indeed, CPT exerted potent growth inhibitory effects on Huh-6, Huh-7, and HCCLM3 cells, but was much less effective on SMMC-7721, PLC/PRF/5, SK-hep-1, QGY-7701, and HepG2 cells. Therefore, we used HCCLM3 and Huh-7 cells in subsequent experiments.

Table 1

IC₅₀ (μM) Values of CPT in 8 Kinds of Human HCC Cells and Hepatocyte Line LO2.

Cell lines	24 hours	48 hours	72 hours
SMMC-7721	＞20	＞20	16.2
PLC/PRF/5	＞20	＞20	10.6
SK-hep-1	＞20	＞20	15.2
QGY-7701	＞20	＞20	18.2
HepG2	＞20	＞20	13.2
Huh-6	13.5	4.3	1.7
HCCLM3	＞20	10.7	8.9
Huh-7	4.2	2.3	1.0
LO2	＞20	＞20	12.6

Abbreviations: CPT, cryptotanshinone; HCC, hepatocellular carcinoma; IC₅₀, half-maximal inhibitory concentration.

To investigate further whether CPT could inhibit the viability of HCCLM3 and Huh-7 cells, we conducted colony formation assays after CPT treatment, which indicated the number of adherent cells that survive and form clones after inoculation. CPT was found to reduce the clone size and clone quantity of HCCLM3 and Huh-7 in a dose-dependent manner (Figure 1(k)); the statistical results of cell colony quantities are shown in Figure 1(l).

CPT Induced Apoptosis in HCCLM3 and Huh-7 Cells

Cell apoptosis exhibits plenty of morphological features, typically including cell shrinkage, nuclear fragmentation, and chromatin condensation, which can be observed using the DNA fluorescent dye Hoechst 33258. ¹⁸ As shown in Figure 2(a), CPT was found to induce apoptosis in both HCCLM3 and Huh-7 cells in a concentration-dependent manner, while bright-blue fluorescent-condensed nuclei and nuclear fragmentation were clearly observed in the CPT treatment groups. To confirm the apoptosis of HCC cells induced by CPT, we next assessed the percent of apoptosis by FCM using an Annexin V-FITC/PI dual-labeling technique. As shown in Figure 2(b), the results further proved that CPT could induce apoptosis of HCCLM3 and Huh-7 cells in a concentration-dependent manner. After treatment with a 20 µM solution for 48 hours, 40.6%, and 10.2% apoptosis was detected for HCCLM3 and Huh-7 cells, respectively (Figure 2(c)).

Figure 2

CPT induced HCCLM3 and Huh-7 cells apoptosis. (a) The fluorescence microscopic appearance of Hoechst 33258 staining nuclei of HCCLM3 and Huh-7 cells with CPT (0-10 μM) for 48 hours (20×). (b) HCCLM3 and Huh-7 cells were incubated with CPT (0-20 μM) for 48 hours, and then were labeled with PI or Annexin V/PI dual-labeling followed by FCM analysis. (c) The proportions of apoptotic cells. (d) Western blot analyses of HCCLM3 and Huh-7 cells treated with different concentrations of CPT (0-20μM) for 48 hours to evaluate proteinexpression of Bcl-2, Bax, cleaved-caspase-3. (e) The percentage of Bax/Bcl-2 ratio; *P < 0.05 vs control group; ***P < 0.001 vs control group. CPT, cryptotanshinone; FCM, flow cytometry; PI, propidium iodide

To clarify further the mechanism of increased apoptosis induced by CPT in HCC cells, we explored Western blot assay to examine cleaved-caspase-3, Bcl-2, and Bax expression levels in HCCLM3 and Huh-7 cells after CPT treatment. As shown in Figure 2(d) and (e), CPT significantly increased the expression of cleaved-caspase-3 and Bax, while decreasing the expression of Bcl-2 in a dose-dependent manner.

CPT Triggered the Imbalance of Mitochondrial Transmembrane Potential (Δψ_m ) and ROS

Aimed at identifying that CPT induces changes in the mitochondrial membrane potential, we evaluated variations in this (ΔΨ_m ) using a mitochondria-specific and voltage-dependent dye, Rh123, by FCM. Rh123 is a mitochondrial-binding specific cationic fluorescent dye that is proportional to the Δψ_m . ¹⁹ As shown in Figure 3(a), CPT treatment led to the loss of Δψ_m potential in HCCLM3 cells while gaining a profit in Huh-7. These results suggested that CPT might tip the balance in the intracellular environment.

Figure 3

CPT induced the variation in the mitochondrial membrane potential (ΔΨ _m ) and the level of ROS in HCCLM3 and Huh-7 cells. (a) HCCLM3 and Huh-7 cells were treated with different concentrations of CPT (0-20 μM) for 48 hours and then stained with 5 μg/ml Rh123 to test the changes of ΔΨ _m by FCM; (b) HCCLM3 and Huh-7 cells were treated with CPT (0-20 μM) for 48 hours followed by analysis of ROS by FCM. CPT, cryptotanshinone; FCM, flow cytometry; ROS, reactive oxygen species.

Mitochondria are the main source of ROS. Therefore, the level of ROS was also measured by FCM using an indicator of DCFH-DA. Similar to the results of Δψ_m potential, we discovered that treatment with CPT lessened ROS accumulation in HCCLM3 cells and multiplied in Huh-7, which can be explicitly observed in Figure 3(b). These results suggest that CPT-induced apoptosis might be via the mitochondrial apoptotic pathway. The changing trend of ROS and Rh123 was different in HCCLM3 and Huh-7 cells, which may indicate that different mechanisms of the therapeutic effect of CPT may be involved for different HCC cells.

CPT Impaired HCCLM3 and Huh-7 Cell Migration and Invasion

To determine whether CPT could inhibit human HCC cell migration and invasion, we carried out wound-healing assay and transwell migration assay with HCCLM3 and Huh-7 cells. As shown in Figure 4(a) and (b), CPT inhibited the migration of both HCCLM3 and Huh-7 cells in a concentration-dependent manner. Furthermore, migration inhibition was observed in the transwell migration assay (Figure 4(c) and (d)). Compared with the control group, CPT could effectively attenuate HCCLM3 and Huh-7 cell migration in a concentration-dependent manner.

Figure 4

CPT inhibited migration and invasion in HCCM3 and Huh-7 cells. (a) HCCLM3 and Huh-7 cells were seeded in 6-well plates. A single scratch was made after the cells grew about 80% confluence. After treatment of CPT (0-10 μM) for 0 hour and 48 hours, the cells were photographed (4×). (b) The migrated cells derived from the same area were quantified. (c) HCCLM3 and Huh-7 cells were seeded in the top chamber of transwell which pretreated with or without Matrigel, after 48 hours treated with CPT (0-10 μM), invaded or migrated cells were fixed, stained, photographed and quantified (20×). (d) Migrated or invaded cell number was counted; *P < 0.05 vs control group; **P < 0.01 vs control group; ***P < 0.001 vs control group. CPT, cryptotanshinone.

We also conducted a Matrigel invasion assay to evaluate the ability of HCCLM3 and Huh-7 cells to invade through Matrigel and the membrane barrier of the transwell. As displayed in Figure 4(c) and (d), in contrast with the control group, the invasion was significantly decreased for both HCCLM3 and Huh-7 cells after CPT treatment.

To further clarify whether p-Stat3, MMP-2, and MMP-9 are closely related to cell migration and invasion, we carried out Western blot analysis. As shown in Figure 5(a) and (b), expression levels of p-Stat3, MMP-2, and MMP-9 showed significant decreases after 48-hour stimulation with CPT, while the expression of TIMP-2, a tissue inhibitor of MMPs, increased quite significantly. These results revealed that CPT is effective in inhibiting the migration and invasion of human HCC cells.

Figure 5

The changes of p-Stat3, STAT3, TIMP-2, MMP-9, and MMP-2 expression. (a) HCCLM3 and Huh-7cells were treated with different concentrations of CPT(0-20μM). After 48 hours, cells were harvested and lysed. Western blot assay was conducted to detect the expression of p-Stat3, STAT3, TIMP-2, MMP-9, and MMP-2, while β-actin served as loading control. (b) The expression of p-Stat3/STAT3, TIMP-2, MMP-9, and MMP-2 were quantified and normalized against β-actin expression; *P < 0.05 vs control group; **P < 0.01 vs controlgroup; ***P < 0.001 vs control group. MMP, matrix metalloproteinases; STAT3, signal transducers and activators of transcription 3; TIMP, tissue inhibitor of metalloproteinases.

Discussion

HCC is one of the most common forms of cancer and a critical cause of cancer mortality globally. Clinical success in the pharmacological treatment of HCC patients has been limited. ²⁰ One of the important features of HCC is high morbidity, mortality, and poor prognosis due to recurrence and metastasis, and so inhibiting the metastasis ability of HCC might be an effective treatment for malignant tumors. ²¹ Therefore, the discovery of novel drugs for the treatment of HCC metastasis is critical.

As an active ingredient of the Chinese traditional herbal Danshen, CPT has been widely studied in a myriad of diseases. ²² A previous study by our team found that CPT could inhibit human melanoma cancer cell viability through apoptosis. ¹² In the present study, we found that CPT significantly inhibited the viabilities of SMMC-7721 and 7 other kinds of human HCC cell lines. Huh-6, Huh-7, and HCCLM3 cells were more sensitive than the other HCC cell lines, and there was little difference between Huh-6 and Huh-7 cells, so we chose Huh-7 and HCCLM3 cells for this study. The current work provided evidence that CPT could inhibit the proliferation of HCCLM3 and Huh-7 cells through apoptosis induction. MTT and colony formation assays indicated that CPT could inhibit HCCLM3 and Huh-7 cell growth and proliferation in a dose-dependent and time-dependent manner.

Apoptosis, also known as programmed cell death, refers to spontaneous and orderly cell death controlled by genes to maintain cellular homeostasis. It involves the activation, expression, and regulation of a series of genes. Furthermore, mitochondria play a central role in the intrinsic apoptosis process via altering the mitochondrial transmembrane potential. Numerous studies have shown that the interaction between proapoptotic and antiapoptotic proteins in the Bcl-2 family of proteins controls the release of cytochrome C from mitochondria and plays an important role in regulating the apoptotic pathway of mitochondria. ^23,24

In this study, apoptotic bodies with bright blue fluorescence were observed by Hoechst 33 258 experiment, while the Annexin V-FITC/PI dual-labeling assay revealed that CPT could distinctly increase the apoptosis rate of human HCC cells at the late phase. In addition, CPT triggered the activation of cleaved-caspase-3 and Bax while inducing the downregulation of Bcl-2. The imbalance of ΔΨ_m could consequently induce the disproportion of intracellular ROS, which indicated that CPT treatment significantly induced apoptosis in human HCC cells via the ROS-medicated mitochondrial apoptotic pathway.

Metastasis is one of the main causes of HCC recurrence and is the primary cause of cancer-related death in the clinic. ²⁵ Cancer liver metastasis is a complex process, including several major steps: microvascular invasion and infiltration; cell survival in the circulation and establishment in the liver; formation of metastatic niches; and expansion of tumor cells. ²⁶ Hence, inhibition of tumor cell migration and invasion might represent a preferred therapeutic strategy for metastatic human HCC. MMPs are a family of zinc-dependent proteolytic enzymes, which have high expression in metastatic carcinoma. ²¹ Especially, the extracellular matrix metalloproteases MMP-9 and MMP-2 are critical for the invasive potential of tumors, as unequivocal data demonstrate that levels of MMP-2 and MMP-9 increase with the progression of liver tumors. ^27,28 In this study, we conducted the wound-healing assay and transwell migration and invasion assays to prove the antimetastasis ability of CPT. Herein, CPT attenuated HCCLM3 and Huh-7 cells migratory and invasive ability via down-regulating the expressions of metastasis-related proteins, especially MMP-2 and MMP-9. Collectively, our study demonstrated that CPT is effective in inhibiting human HCC cell migration and invasion.

In conclusion, our study found that CPT could inhibit the growth of HCC cells, promote the apoptosis of HCCLM3 and Huh-7 cells through the mitochondrial pathway, and inhibit migration and invasion of HCC cells by inhibiting the expression of metastasis-related proteins (Figure 6). These data strongly suggest the value of a further study on the application of CPT as a potential treatment for HCC.

Figure 6

Schematic presentation of the signaling pathways in HCC cells affected by CPT.