Shi-pi-xiao-ji formula suppresses hepatocellular carcinoma by reducing cellular stiffness through upregulation of acetyl-coA acetyltransferase 1

Abstract

BACKGROUND

Previous studies have shown that the Shi-pi-xiao-ji (SPXJ) herbal decoction formula is effective in suppressing hepatocellular carcinoma (HCC), but the underlying mechanisms are not known. Therefore, this study investigated whether the antitumor effects of the SPXJ formula in treating HCC were mediated by acetyl-coA acetyltransferase 1 (ACAT1)-regulated cellular stiffness. Through a series of experiments, we concluded that SPXJ inhibits the progression of HCC by upregulating the expression level of ACAT1, lowering the level of cholesterol in the cell membrane, and altering the cellular stiffness, which provides a new idea for the research of traditional Chinese medicine against HCC.

AIM

To investigate the anti-tumor effects of the SPXJ formula on the malignant progression of HCC.

METHODS

HCC cells were cultured in vitro with SPXJ-containing serum prepared by injecting SPXJ formula into wild-type mice. The apoptotic rate and proliferative, invasive, and migratory abilities of control and SPXJ-treated HCC cells were compared. Atomic force microscopy was used to determine the cell surface morphology and the Young’s modulus values of the control and SPXJ-treated HCC cells. Plasma membrane cholesterol levels in HCC cells were detected using the Amplex Red cholesterol detection kit. ACAT1 protein levels were estimated using western blotting.

RESULTS

Compared with the vehicle group, SPXJ serum considerably reduced proliferation of HCC cells, increased stiffness and apoptosis of HCC cells, inhibited migration and invasion of HCC cells, decreased plasma membrane cholesterol levels, and upregulated ACAT1 protein levels. However, treatment of HCC cells with the water-soluble cholesterol promoted proliferation, migration, and invasion of HCC cells as well as decreased cell stiffness and plasma membrane cholesterol levels, but did not alter the apoptotic rate and ACAT1 protein expression levels compared with the vehicle control.

CONCLUSION

SPXJ formula inhibited proliferation, invasion, and migration of HCC cells by decreasing plasma membrane cholesterol levels and altering cellular stiffness through upregulation of ACAT1 protein expression.

Key Words: Shi-pi-xiao-ji formula, Hepatocellular carcinoma, Cellular stiffness, Plasma membrane cholesterol level, Acetyl-coA acetyltransferase 1

Core Tip: Cellular stiffness is a key feature of cellular biomechanics and is determined by cholesterol levels in the plasma membrane. Traditional Chinese medicine (TCM) has demonstrated good anti-tumor effects, but underlying mechanisms are not well characterized. Herein, we investigated the effects of the Shi-pi-xiao-ji (SPXJ) herbal formula on the stiffness of hepatocellular carcinoma (HCC) cells. The SPXJ formula altered the stiffness of HCC cells by decreasing plasma membrane cholesterol levels through upregulation of acetyl-coA acetyltransferase 1. Subsequently, this inhibited the invasion and metastasis of HCC cells. Our study provides a scientific basis for the clinical use of TCM preparations in the treatment of HCC.

INTRODUCTION

Primary liver cancer (PLC) is a serious malignancy characterized by considerable histological and biological heterogeneity. According to the GLOBOCAN 2020 data, 910000 new cases of liver cancer and 830000 PLC-related deaths were reported worldwide[1]. PLC includes malignant tumors originating from hepatocytes or intrahepatic bile duct epithelial cells and is classified into the following three distinct pathological types: Hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined HCC-ICC. HCC accounts for approximately 90% of the PLC cases. HCC is a significant global health challenge because of high prevalence and mortality rates, with an estimated 5-year survival rate of only 18%[2]. PLC is a highly prevalent malignant tumor in China with 45.3% of new cases and 47.1% deaths; moreover, PLC cases in China account for approximately half of the global incidence rates (n = 389000; fourth rank) and deaths (n = 336000; second rank)[3]. The median survival rate for liver cancer in China is approximately 23 months[4], and for advanced liver cancer, it is only 6 months[1,5]. Therefore, PLC is a remarkable burden for both patients and society. The treatment modalities for PLC vary depending on the disease stage. Available treatments include resection, radiofrequency ablation (RFA), interventional embolization, radiotherapy, systemic chemotherapy, as well as molecular targeted therapy and immunotherapy. However, PLC is associated with insidious onset, high malignancy, and rapid progression. Therefore, a considerable number of patients are diagnosed at intermediate or advanced stages of PLC and are not amenable to surgical intervention. Despite the approval and clinical testing of numerous drugs for advanced HCC, the median progression-free survival and overall survival rates remain disheartening[6]. The diagnostic and therapeutic landscape for PLC is therefore challenging because of the severity and diversity of the disease.

The rapid progression, recurrence, and metastatic potential of cancer is mainly influenced by the proliferative, invasive, and migratory abilities of the cancer cells. Therefore, suppressing proliferation, invasion, and migration of the HCC cells is a major strategy for inhibiting HCC progression. The migration and invasiveness of HCC cells involves changes in their biomechanical properties to facilitate intricate cellular movements and deformations. Cellular biomechanics plays a pivotal role in the behavior of cancer cells. Cellular stiffness is a key feature that defines the biomechanical properties of cancer cells. Cellular stiffness is intricately related with the motility and metastatic potential of cancer cells[7-9]. The cholesterol concentration in the cell membrane is one of the main determinants of cellular stiffness. Depletion of cholesterol levels in the cell membrane of tumor cells is associated with increased cellular stiffness[10]. Acetyl-coA acetyltransferase 1 (ACAT1) (also named acyl-coenzyme A: Cholesterol acyltransferase) is an enzyme that transfers long-chain fatty acids to cholesterol to form cholesteryl esters, which subsequently coalesce and form cytosolic lipid droplets[11]. Therefore, ACAT1 regulates the cholesterol composition of the cell membrane.

The SPXJ formula contains 15 herbs, namely, Hedysarum multijugum Maxi. (Huangqi), Panax Ginseng C. A. Mey. (Renshen), Poria Cocos (Schw.) Wolf. (Fuling), Fructus Ligustri Lucidi (Nvzhenzi), Epimrdii Herba (Yinyanghuo), Salviae Chinensis Herba (Shijianchuan), Persicae Semen (Taoren), Eupolyphaga steleophaga (Tubeimu), Akebiae Frucyus (Yuzhizi), Curcumae Radix (Yujin), Pinelliae rhizoma praeparatum (Fabanxia), Scutellariae Barbatae Herba (Banzhilian), Solanum Nigrum Linn. (Longkui), Gecko (Bihu), Gleditsiae Spina (Zaojiaoci). SPXJ formula is rich in ginsenosides[12,13], polysaccharides[14,15], curcumin[16-18], and various other anti-tumor active ingredients. A previous study reported that the SPXJ formula suppressed the growth and progression of HCC cells[19-21], specifically by suppressing HepG2 cells[22]. Furthermore, individual herbs used in the preparation of SPXJ formula regulate energy metabolism and apoptosis in HCC cells. However, the specific effects of these components on the biomechanical properties of HCC cells are unclear.

In this study, we investigated whether the anti-tumor effects of the SPXJ formula in the HCC were mediated by ACAT1-regulated alterations in plasma membrane cholesterol levels and cellular stiffness. The main goal was to establish a scientific basis for the use of traditional Chinese medicine (TCM) formulas in the treatment of PLC patients.

MATERIALS AND METHODS

Materials and antibodies

Methyl-β-cyclodextrin (MβCD, Cat. No. GC32697) was purchased from Good Laboratory Practice Bioscience (Montclair, United States). Water-soluble cholesterol (Chol, Cat. No. SLCP6290) was purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified eagle medium (DMEM, Cat. No. PM15210), phosphate-buffered saline (PBS, Cat. No. PB180327), fetal bovine serum (FBS, Cat. No. 164210), penicillin-streptomycin solution (Cat. No. PB180120) were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Trypsin (0.25%) was purchased from ECTOP. Matrigel (Cat. No. 356234) was purchased from corning (New York, United States). Amplex red cholesterol assay kit (Cat. No. 2649096) was purchased from ThermoFisher Scientific (Massachusetts, United States). RIPA lysis buffer (Strong) (Cat. No. CW2333) was purchased from CWBIO (Beijing, China). BCA protein concentration measurement Kit (Cat. No. P0010) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). ACAT1 polyclonal antibody (Cat. No. GB112323), Recombinant anti - beta actin antibody (Cat. No. GB15003), HRP-conjugated goat anti-rabbit IgG (H + L) (Cat. No. GB23303) were purchased from Servicebio Technology Co., Ltd. (Wuhan, China).

Preparation for SPXJ formula

The SPXJ formula contained 15 herbs that were purchased from the Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine (Changsha, China). The botanical composition of the SPXJ formula is listed in Table 1. The herbs were soaked in 5000 mL of water for 30 min at room temperature and then concentrated to 2000 mL by boiling for approximately 30 min. Then, the liquid herbal extract was harvested by filtration. The remaining herbal components were again extracted by boiling with water for another 30 min. Again, the liquid herbal extract was collected and combined with the previously collected liquid herbal extract. The liquid herbal extract was concentrated to 500 mL. The final preparation was sealed and stored at -20 °C. The SPXJ formula was maintained at 4 °C during use.

Table 1 The composition of Shi-pi-xiao-ji formula.

No.	Chinese Pinyin name	Latin scientific name	Plant parts	Amount (g)
1	Huangqi	Hedysarum multijugum Maxi.	Root	15
2	Renshen	Panax Ginseng C. A. Mey.	Root	5
3	Fuling	Poria Cocos (Schw.) Wolf.	Mycorrhizae	15
4	Nvzhenzi	Fructus Ligustri Lucidi	Fruit	9
5	Yinyanghuo	Epmrdii Herba	Leaf	5
6	Shijianchuan	Salviae Chinensis Herba	Leaf	15
7	Taoren	Persicae Semen	Seed	5
8	Tubiechong	Eupolyphaga steleophaga	Whole worm	5
9	Yuzhizi	Akebiae Frucyus	Fruit	9
10	Yujin	Curcumae Radix	Root	9
11	Fabanxia	Pinelliae rhizoma praeparatum	Root	5
12	Banzhilian	Scutellariae Barbatae Herba	Whole plant	15
13	Longkui	Solanum Nigrum Linn.	Whole plant	15
14	Bihu	Gecko	Whole worm	5
15	Zaojiaoci	Gleditsiae Spina	Thorn	6

Animals

We purchased twenty 5-6 wk-old male SD rats (SPF grade; 180-220 g weight) from the Hunan Silaikejingda Experimental Animal Co., Ltd (Changsha, China) (quality certificate number: No. 4307272211011626852). The rats were housed in the Animal Experimental Center of the Hunan University of Chinese Medicine under a 12 h light/dark cycle with ad libitum access to food and water.

Preparation of SPXJ drug-containing serum

Based on the equivalent dose ratios converted to body surface area between humans and rats[23], according to the conversion of 3 times the daily dose of 70 kg adults, the drug group was given 37.26 g/kg/d by gavage with the Realizing the Spleen and Eliminating Accumulation Drink, and the control group was given 10 mL/kg/d by gavage with drinking water. The SPXJ group rats were administered with 2 mL of the prepared SPXJ formula via oral gavage once daily for 7 d. The control group was administered 2 mL of drinking water. One hour after the last intragastric administration, the rats were anesthetized with isoflurane, the abdominal aorta was cut, and the whole blood was collected. The blood was let to stand at room temperature for 1 h. Then, it was centrifuged at 3000 rpm for 15 min and the serum was harvested. The serum samples from each group were pooled, inactivated in a 56 °C water bath for 30 min, passed through a 0.22-μm filter, and stored at -20 °C for later use.

Cell line and culture

The MHCC97H (SNL-401) and HepG2 (SNL-083) human HCC cell lines were purchased from the Sunncell Biotechnology Co., Ltd (Wuhan, China). The cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37 °C in a 5% CO₂ water jacket incubator (Thermo Fisher Scientific). The cells at 5^th passage were used for all the experimental procedures.

Experimental groups and treatment

The logarithmically growing MHCC97H and HepG2 cells were divided into four distinct groups: Vehicle, SPXJ-serum, MβCD, and Chol. The cells in the SPXJ-serum group were cultured in medium containing 10% SPXJ drug-containing serum for 24 h or 48 h. The cells in the MβCD group were cultured in medium supplemented with 5 mmol/L MβCD for 24 h or 48 h. The cells in the Chol group were cultured in medium containing 1 mmol/L water-soluble cholesterol for 24 h or 48 h.

Colony formation assay

MHCC97H and HepG2 cells were seeded in 6-well plates at a density of 800 cells/well and cultured for 24 h. Subsequently, after removing the media, the cells were incubated with fresh growth medium containing different drugs and cultured for a week. The culture medium was then discarded. The cells were then fixed for 15 min with 1 mL of 4% paraformaldehyde. The colonies were stained with 1 mL of 1% crystal violet staining solution for 15 min at room temperature. Then, the wells were washed with running water and dried. The stained cells in the 6-well plates were photographed, and colonies with more than 50 cells were counted.

Wound healing assay

MHCC97H and HepG2 cells were seeded in 6-well plates at a density of 2 × 10⁴ cells/well and cultured until a confluent monolayer was formed for 24 h. A clean scratch wound was then generated at the center of the monolayer in each well with a 200 μL pipette. Subsequently, floating cells were removed by washing with PBS. Then, the monolayers of cells were incubated with fresh DMEM medium supplemented with 10% FBS and specific drugs (SPXJ-serum, MβCD, and Chol). The 6-well plates were incubated in a humidified incubator at 37 °C and 5% CO₂. The wounds were photographed at predetermined time intervals (24 h and 48 h). The migration rate was determined using the Image J software using the following formula: Migration (%) = (initial scratch width - final scratch width)/initial scratch width × 100%.

Transwell assay

The upper chambers of the Transwell were coated with the diluted matrix Matrigel (1:7). MHCC97H and HepG2 cells were resuspended in serum-free DMEM medium and seeded into the upper chambers of the Transwell at a concentration of 1 × 10⁴ cells per 100 μL. The lower chambers of the Transwell were filled with DMEM medium supplemented with 10% FBS, which acted as a chemoattractant for the cells in the upper chamber. Subsequently, various drugs were added into the upper chambers. Transwell chambers were then incubated for 48 h in a humidified incubator maintained at 37 °C and 5% CO₂. The Matrigel and the non-invading cells on the upper surface of the membrane were gently removed using a cotton swab. The invading cells on the lower surface of the Transwell membrane were fixed, stained, visualized, and photographed under a light microscope. Image J software was used to count the number of invading cells in each group.

Cellular apoptosis assay

The apoptotic cells in each group were estimated using the apoptosis detection kit according to the manufacturer’s instructions. MHCC97H and HepG2 cells were digested with EDTA-free trypsin, centrifuged at 1500 rpm for 5 min at 4 °C, and collected. The cells were washed twice with precooled PBS and centrifuged at 1500 rpm for 5 min. The cells [(1-5) × 10⁵ cells per group] were resuspended in 500 μL of 1 × binding buffer. Then, the cells were stained by adding 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI)-staining solution to the cell suspension, mixing gently, and incubating in the dark at room temperature for 10 min. The stained cells were then analyzed by flow cytometry within 1 h. The apoptosis rate was calculated with the following formula: (number of apoptotic cells/total number of cells observed) ×100%.

Atomic force microscope

MHCC97H and HepG2 cells were seeded at a density of 2 × 10⁴ cells/well in 24-well plates and cultured for 24 h to allow the formation of a confluent monolayer. The medium was then removed and various drugs were added. Subsequently, the cells were washed twice with PBS and fixed with 1% glutaraldehyde for 5 min. The fixed cells were washed twice with PBS. The Young’s modulus values of the samples were determined under an atomic force microscope (AFM) after natural air drying by setting the spring constant of the cantilever at 0.5 N. The tips were cleaned with ethanol and UV light was used after each force map to remove the contaminations. The topography and Young’s modulus images were recorded. Young’s modulus maps were generated with a scan area of 10 micrometer × 10 micrometer with 30 × 30 force-indentation curves to determine the Young’s modulus values for the cells from distinct groups.

Quantification of plasma membrane cholesterol levels

The total cellular cholesterol level was first quantified using the Amplex red cholesterol assay kit. MHCC97H and HepG2 cells were fixed with glutaraldehyde (0.1% in PBS) and the total cholesterol was extracted by sonication with the methanol/chloroform mixture (1:2, v/v). After removing the organic solvent under vacuum, the cholesterol level was quantified using the Amplex red cholesterol assay kit according to the manufacturer’s instructions. To quantify the intracellular cholesterol levels, the fixed cancer cells were treated with cholesterol oxidase (2 U/mL in PBS) to oxidize the plasma membrane cholesterol before extraction. The plasma membrane cholesterol levels were calculated by subtracting the intracellular cholesterol levels from the total cellular cholesterol levels as previously described[24].

Western blotting

Total protein extracts were prepared from the MHCC97H and HepG2 cells by incubating with the RIPA lysis buffer at 4 °C for 30 min. The cell extracts were centrifuged at 14000 × g for 10 min at 4 °C and the supernatant was harvested. The protein concentration was measured by the BCA method. The protein concentrations of the samples were adjusted to 1.5 μg of total protein per microliter with the 5 × SDS loading buffer. The samples were denatured at 100 °C for 10 min. The samples were let to cool before use. SDS-PAGE electrophoresis was performed by loading 20 μL of each protein sample (30 μg protein per sample). The separated proteins were transferred from SDS polyacrylamide gels onto the nitrocellulose membranes. These membranes were blocked with 5% nonfat milk at room temperature for 1 h. Then, they were incubated overnight at 4 °C with primary antibodies such as anti-ACAT1 (1:1000), and anti-ACTIN (1:5000). The membranes were then washed three times with TBST (10 min each) and subsequently incubated with the HRP-conjugated secondary antibodies (1:10000) for 60 min. The membranes were washed thrice with TBST and the protein bands were visualized using the ECL detection system. The relative protein expression levels were then quantified using the Image J software.

Statistical analysis

Statistical analysis was performed using the SPSS 22.0 statistical software. The data with normal distribution were expressed as means ± SE with a sample size of n =3. The differences between multiple groups were analyzed using the One-Way Analysis of Variance when the data satisfied the assumptions of normality and homogeneity of variances. Two-by-two comparisons between multiple groups were performed using the LSD-t test. However, if the assumption of homogeneity of variances was not met, the Welch test was used to compare the groups. P value < 0.05 was determined as statistically significant.

RESULTS

SPXJ-serum treatment induces remarkable changes in HCC cell morphology

The morphological changes in the vehicle- and drug-treated MHCC97H and HepG2 cells were analyzed using an inverted microscope (Figure 1). The cells in the vehicle group exhibited an epithelioid monolayer arrangement, and were characterized by irregular polygonal shapes, close adherence to the substrate, uniform size, and well-defined boundaries. In contrast, cells in the MβCD group were characterized by elongated or spindle-shaped morphology, uneven size, and indistinct margins in some cells (highlighted by orange arrows). The cells treated with the SPXJ-serum exhibited a more rounded shape and were accompanied by increased cellular debris in the culture fluid. The cells in the Chol group displayed irregular polygonal shape with clear and sharp edges. These observations suggested that treatment with the SPXJ formula induced remarkable changes in cell morphology.

Figure 1 Representative images show the morphology of the vehicle, Shi-pi-xiao-ji-serum, methyl-β-cyclodextrin, and water-soluble cholesterol groups of MHCC97H and HepG2 cells. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

SPXJ inhibits proliferation of HCC cells

Colony formation assay was performed to determine the effects of SPXJ on the proliferation of MHCC97H and HepG2 cells. In comparison with the vehicle group, the number and size of colonies were considerably reduced in the SPXJ-serum group (Figure 2). The number of colonies were considerably higher in the Chol group compared with the vehicle group. However, the number of colonies in the MβCD group did not show statistically significant differences with the vehicle group. These data suggested that the SPXJ formula inhibited the proliferation of HCC cells.

Figure 2 Representative images and statistical results of the colony formation assay in the vehicle, Shi-pi-xiao-ji-serum, methyl-β-cyclodextrin, and water-soluble cholesterol groups of MHCC97H and HepG2 cells. A: Representative images; B: Representative statistical results. The results are expressed as means ± SEM (n = 3). ^aP < 0.05, ^bP < 0.01, ns: P > 0.05. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

SPXJ inhibits migration and invasion of HCC cells

The effects of the SPXJ formula on the migration and invasiveness of HCC cells were assessed using wounding healing and Transwell assays, respectively. The results of the wounding healing assay are shown in Figure 3A-D. At 24 h post-treatment, SPXJ-serum-treated MHCC97H cells did not show notable differences in the migration ability compared with the vehicle group, but the migration of SPXJ-serum treated HepG2 cells was considerably reduced. However, at 48 h, migration ability of the SPXJ-serum-treated MHCC97H and HepG2 cells was considerably reduced compared with the vehicle group. Furthermore, MβCD treatment remarkably reduced migration ability of both MHCC97H and HepG2 cells, whereas treatment with water-soluble cholesterol significantly increased the migration ability of MHCC97H and HepG2 cells. Moreover, the effects of different drug treatments were proportional to the duration of treatment. Transwell assay results are shown in Figure 3E and F. In comparison with the vehicle group, the invasiveness of SPXJ-serum- or MβCD- treated MHCC97H and HepG2 cells was significantly reduced. Conversely, the invasiveness of the water-soluble cholesterol-treated MHCC97H and HepG2 cells was significantly higher than the vehicle group. These findings demonstrated that the SPXJ formula inhibited in vitro migration and invasion of the MHCC97H and HepG2 cells.

Figure 3 Representative images and statistical results of wounding healing and transwell assays. A and B: Representative images and statistical results of the wounding healing assay in the vehicle, Shi-pi-xiao-ji (SPXJ)-serum, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol (Chol) groups of MHCCC97H cells; C and D: Representative images and statistical results of wounding healing assay in the vehicle, SPXJ-serum, MβCD, and Chol groups of HepG2 cells; E and F: Representative images and statistical results of the transwell assay in the vehicle, SPXJ-serum, MβCD, and Chol groups of hepatocellular carcinoma cells. The results are expressed as means ± SEM (n= 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ns: P > 0.05. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

SPXJ induces apoptosis of HCC cells

Flow cytometry analysis of annexin V/PI-stained cells was used to determine whether SPXJ formula induced HCC cell death (Figure 4). In comparison with the vehicle group, the proportion of apoptotic cells were significantly higher in the SPXJ-serum-treated MHCC97H cells (15.13% vs 35.31%). Furthermore, the proportion of apoptotic cells were significantly higher in the SPXJ-serum-treated HepG2 cells compared with the vehicle-treated HepG2 cells (19.37% vs 11.44%). The proportion of apoptotic cells were significantly higher in the Chol group compared with the MβCD group. These findings demonstrated that treatment with the SPXJ-serum induced apoptosis in the HCC cells.

Figure 4 Flow cytometry results show the percentage of apoptotic hepatocellular carcinoma cells in the vehicle, Shi-pi-xiao-ji-serum, methyl-β-cyclodextrin, and water-soluble cholesterol groups of MHCC97H cells and HepG2 cells based on the annexin V/PI double-staining. A and B: Flow cytometry results show the percentage of apoptotic hepatocellular carcinoma (HCC) cells in the vehicle, Shi-pi-xiao-ji (SPXJ)-serum, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol (Chol) groups of MHCC97H cells based on the annexin V/PI double-staining; C and D: Flow cytometry results show the percentage of apoptotic HCC cells in the vehicle, SPXJ-serum, MβCD, and Chol groups of HepG2 cells based on the annexin V/PI double-staining. The results are expressed as means ± SEM (n= 3). ^aP < 0.01, ^bP < 0.001 and ns: P > 0.05. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol; PI: Propidium iodide.

SPXJ decreases stiffness of HCC cells

The effects of the SPXJ formula on the stiffness of HCC cells was assessed using AFM. The representative AFM images and the Young’s modulus values for the HCC cells in all the groups are shown in Figure 5. In comparison with the vehicle group, the Young’s modulus values were significantly higher in the SPXJ-serum and MβCD group cells, but considerably decreased in the Chol group. The brightness in the AFM images corresponds with the height and the brightest area denotes the nucleus. The cell surface in the vehicle group displayed holes, regular and flat surface, and brighter morphology. In comparison with the vehicle group and SPXJ-serum group, cells in the Chol group showed remarkable alterations in the cell surface morphology, including presence of large holes with varying depths on the cell surface (white arrow in the figure). Conversely, the MβCD group displayed a flat and smooth cell surface and did not show any obvious holes. These data suggested that SPXJ treatment altered the stiffness of the HCC cells.

Figure 5 Representative atomic force microscope images and Young’s modulus values. A and B: Representative atomic force microscope (AFM) images (A) and Young’s modulus values (B) of MHCC97H cells in the vehicle, Shi-pi-xiao-ji (SPXJ)-serum, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol (Chol) groups; C and D: Representative AFM images (C) and Young’s modulus values (D) of HepG2 cells in the vehicle, SPXJ-serum, MβCD, and Chol groups. The results are expressed as means ± SEM (n= 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

SPXJ increases cellular stiffness by lowering plasma membrane cholesterol levels

Cholesterol-rich domains in the cell membranes regulate cellular stiffness. Therefore, we analyzed the effects of SPXJ-serum treatment on the cholesterol levels in the plasma membrane of the HCC cells. Amplex red cholesterol assay results are shown in Figure 6. SPXJ-serum and Chol groups showed significantly higher cholesterol levels in the plasma membrane of the HCC cells compared with the vehicle group, but the levels of plasma membrane cholesterol were significantly lower in the MβCD group. This demonstrated that SPXJ increased stiffness in the HCC cells by decreasing plasma membrane cholesterol levels.

Figure 6 The total plasma membrane cholesterol levels in the vehicle, Shi-pi-xiao-ji-serum, methyl-β-cyclodextrin, and water-soluble cholesterol groups of MHCC97H and HepG2 cells. The results are expressed as means ± SEM (n= 3). ^cP < 0.001. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol.

SPXJ reduces plasma membrane cholesterol levels by increasing ACAT1 expression levels

ACAT1 is an enzyme that regulates formation of cholesteryl esters by transferring a long-chain fatty acid to cholesterol. Thus, ACAT1 regulates the levels of cholesterol in the cell membrane. Therefore, we performed western blotting to determine if the SPXJ formula decreased the stiffness of HCC cells by increasing ACAT1 protein expression levels. Western blotting results demonstrated that treatment with SPXJ-serum significantly upregulated ACAT1 protein levels while concurrently decreasing cholesterol levels in the plasma membrane (Figure 7). This, in turn, increased the stiffness of HCC cells and inhibited their migration and invasion. However, treatment of HCC cells with the cholesterol-depleting agent MβCD or water-soluble cholesterol did not show any statistically significant changes on the ACAT1 protein expression levels.

Figure 7 Western blot analysis of acetyl-coA acetyltransferase 1 protein expression levels. A: Representative image shows the expression of acetyl-coA acetyltransferase 1 (ACAT1) protein in vehicle, Shi-pi-xiao-ji (SPXJ)-serum, methyl-β-cyclodextrin (MβCD), and water-soluble cholesterol (Chol) groups of MHCC97H and HepG2 cells. Actin was used as loading control; B: Histogram plots show the relative expression levels of ACAT1 protein in the vehicle, SPXJ-serum, MβCD, and Chol groups of MHCC97H and HepG2 cells. The results are expressed as means ± SEM (n= 3). ^aP < 0.01, ns: P > 0.05. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; Chol: Water-soluble cholesterol; ACAT1: Acetyl-coA acetyltransferase 1.

DISCUSSION

PLC is the fourth leading cause of cancer-related deaths globally[25,26]. Therefore, PLC is a considerable public health challenge. The morbidity and mortality rates of patients with PLC have increased considerably over the past decades. According to the World Health Organization data, an estimated 1276679 individuals are estimated to die because of liver cancer in the year 2040. The incidence and mortality rates of PLC show substantial regional variations with countries in East Asia and sub-Saharan Africa reporting the highest incidence rates. The incidence rate of PLC is high in China with an estimated 18.3 cases per 100000 individuals[27,28]. Patients with early or intermediate-stage HCC are treated with localized locoregional therapies, including RFA, resection, transplantation, percutaneous ethanol injection, or transcatheter arterial chemoembolization. Furthermore, patients with HCC can be treated with systemic approaches such as chemotherapy, molecular targeted therapy, and immunotherapy. Despite promising developments in drug discovery, the prognosis of HCC remains poor. The median progression-free survival and overall survival rates of intermediate and advanced stage in patients with HCC remain considerably low. The diagnosis and treatment of PLC, especially HCC, remains a substantial challenge.

TCM has emerged as an effective alternative treatment for HCC patients with minimal side effects[29]. Several in vivo and in vitro experiments have shown that Chinese herbal formulas are based on invigorating qi, strengthening the spleen, tonifying the kidneys, and removing toxins and blood stasis. Therefore, TCM treatments considerably reduces proliferation, survival, and progression of HCC cells by regulating key signaling pathways such as PI3K/Akt, β-catenin, MAPK3, and RHOA[30-32]. SPXJ formula has been shown to stabilize tumors, improve patients’ quality of life, and resist recurrence and metastasis in the clinical treatment of liver cancer[33,34]. In this study, we employed colony formation, wound healing, Transwell, and apoptosis assays to investigate the effects of the SPXJ formula on the HCC cells. The results demonstrated that the SPXJ formula effectively inhibited the proliferation, migration, and invasion of HCC cells. Moreover, the SPXJ formula significantly increased apoptosis of HCC cells. These results corroborated previous findings by our group[22]. Our experiments also demonstrated that the number of colonies were higher in the Chol group. This may be caused by cholesterol promoting lipid metabolism in the tumor cells and facilitating their proliferation[35-37].

Previous and current research into the mechanisms of tumor metastasis have focused on processes such as integrin-mediated cell adhesion, epithelial-mesenchymal transition, tumor angiogenesis, lymphangiogenesis, and the hypoxic microenvironment. However, the effects of mechanical forces on the tumor invasion of the extracellular matrix and tumor cell migration in the matrix are not well characterized. The biomechanical properties of cells influence cytoplasmic membrane fluidity, cell stiffness, adhesion, and structural composition of the cytoskeleton. Therefore, there is a greater need to investigate the mechanical aspects that influence tumor metastasis[38]. Cellular biomechanics is intricately linked with various cell behaviors, including division, differentiation, migration, and apoptosis. Cellular biomechanics plays a pivotal role in tumor metastasis. During the process of metastasis, tumor cells rely on movement and deformation for metastasis, invasion, and cell-cell adhesion. Therefore, understanding the biomechanics of these processes is crucial for gaining insights into the mechanisms underlying tumor metastasis and may also contribute to the development of targeted therapeutic strategies. Our data demonstrated that treatment with SPXJ-serum significantly inhibited migration of the HCC cells, with noticeable reduced rate of migration to the distant sites. Furthermore, treatment with SPXJ-serum reduced invasion of HCC cells into the Matrigel. This suggested that SPXJ may inhibit HCC cells from invading the stroma. The effects in HCC cells treated with SPXJ were similar to the effects observed in the MβCD-treated HCC cells. Conversely, HCC cells in the Chol group showed significantly higher migration and invasion abilities. These data were also in agreement with the morphological changes observed using the AFM. The SPXJ-treated HCC cells showed round morphology. Furthermore, increased cellular debris was observed in the culture medium of the SPXJ-treated HCC cells. In contrast, HCC cells in the MβCD group exhibited significantly elongated cell morphology and blurred edges. HCC cells in the Chol group did not show significant alterations in the cell morphology compared with the control group, except for the presence of slightly sharper edges.

The capacity of tumor cells to move and deform is associated with their mechanical properties. The stiffness of a tumor cell is a key feature that modulates essential functions such as cell division, differentiation, migration, and apoptosis. Cellular stiffness is associated with the cell membrane and subcellular components, including the cytoskeleton, nucleus, and other organelles. Therefore, understanding and characterizing these mechanical properties will provide valuable insights into the behavior and functions of tumor cells. The cytoskeleton is primarily composed of microtubules, actin filaments, and intermediate fibers[39]. It represents the spatial architecture of a cell and acts as a connecting bridge between the cell and the external environment. The cytoskeleton plays a crucial role in facilitating cell migration and deformation[40]. Furthermore, the cytoskeleton contributes to overall plasticity of a cell[41]. Several studies have demonstrated a strong association between aberrant changes in cellular stiffness and cancer development[42]. Cancer cells exhibited significantly reduced elastic modulus values compared to the healthy cells[41]. The relative softness of the invasive tumor cells decreased their adhesion to the extracellular matrix and augmented their ability to detach from the primary tumor site. Young’s modulus values are used to quantify cell stiffness, and indicate the extent of deformation in the tumor cells[38,43]. Lekka et al[44] performed AFM and reported that the reduced stiffness of highly invasive ovarian cancer cells (HeyA8) cells was associated with the remodeling of the actin cytoskeleton. Therefore, AFM was used in this study to determine changes in the stiffness of HCC cells. In comparison with the vehicle group, HCC cells in the SPXJ-serum group demonstrated regular cell surfaces without noticeable pores and higher Young’s modulus values, both of which were indicative of increased cell stiffness. HCC cells in the MβCD group exhibited bright and regular cell areas. They also showed the highest Young’s modulus values among different treatment groups. Conversely, HCC cells in the Chol group displayed blurred edges and pores. They also showed the lowest Young’s modulus values among all treatment groups, thereby suggesting reduced cell stiffness.

In mammalian cells, cholesterol is a crucial component of the cell membranes[45,46], and constitutes up to 50% of the plasma membrane lipids[11]. It is involved in various cellular functions[46-49]. Cholesterol modulates membrane fluidity by modifying the permeability barrier properties of the lipid bilayers[50]. Cholesterol also regulates turgidity of the cell membrane[51] by influencing various physical mechanisms that are necessary for membrane homeostasis[52]. Several studies have suggested that cholesterol-rich membrane domains that are formed in the plasma membrane play a crucial role in regulating cellular stiffness. A previous study investigated how the biomechanical properties of cancer cells influence T-cell-mediated cytotoxicity and adoptive T-cell immunotherapy, and reported that malignant transformation and tumor progression was associated with the softening of the cancer cells. The plasma membranes of soft cancer cells are enriched in cholesterol. Therefore, stiffening of cancer cells by cholesterol depletion enhanced T-cell cytotoxicity and the efficacy of adoptive T-cells therapy against solid tumors in mice[10]. In the model membranes, cholesterol content influences fundamental mechanical parameters such as bending stiffness and modulus of elasticity[53] and the resistance of the membrane to stress-induced rupture such as increased line tension[53,54]. Biswas et al[55] demonstrated that cholesterol-depleted cells were prone to rupture because of increased membrane tension and its variability. In conjunction with the findings from previous studies, the regular cell morphology and increased invasive and migratory abilities of HCC cells in the Chol group may be linked to elevated cholesterol levels in the cell membrane facilitated by exogenous water-soluble cholesterol, thereby reducing cellular stiffness. Conversely, the diminished invasive and migratory abilities of HCC cells in the MβCD group may be attributed to the depletion of cholesterol levels in the cell membrane by MβCD. The effects of SPXJ formula on the HCC cells may also be attributed to changes in cholesterol levels in the cell membrane. This was verified by estimating cholesterol levels in the plasma membranes of HCC cells from distinct groups.

ACAT gene was first identified in 1993[56]. The ACAT gene family consists of ACAT1, ACAT2[57-59], and acyl-CoA: Diacylglycerol acyltransferase 1[60]. ACAT1 accounts for 80% of the total ACAT enzyme activity measured in vitro, and is expressed in many tissues and cell types, including hepatocytes and Kupffer cells of the liver, adrenal glands, neurons, and macrophages[61,62]. ACAT1 and ACAT2 are integral membrane proteins. ACAT1 and ACAT2 are not expressed simultaneously under physiological settings in the same cell type in any tissue[59,62]. As a cholesterol-metabolizing enzyme, ACAT1 forms cholesteryl esters by transferring long-chain fatty acids to cholesterol. We investigated whether the effects of the SPXJ formula on the stiffness of HCC cells were linked to changes in the ACAT1 protein expression levels. Western blotting experiments demonstrated that SPXJ-serum significantly increased ACAT1 protein expression levels compared to the vehicle group. Furthermore, depletion of cholesterol levels with MβCD or addition of exogenous cholesterol did not alter the protein expression levels of ACAT1. Previous studies have shown that the activity of ACAT2 was highly variable but the activity of ACAT was relatively constant[63].

The present study is a preliminary investigation into the role of SPXJ formula in altering cellular stiffness by regulating the expression levels of ATAC1 through in vitro experiments. It is not yet possible to attribute the role to any single component of SPXJ formula and the underlying molecular mechanisms are not yet clear. In the future, we plan to perform high-throughput screening and immunoprecipitation experiments to confirm the underlying mechanisms by which SPXJ formula modulates stiffness of tumor cells, including HCC.

CONCLUSION

In summary, our findings demonstrated that the SPXJ formula inhibited HCC invasion and metastasis by increasing ACAT1 protein expression levels and cellular stiffness by reducing cholesterol levels in the plasma membranes. This suggested that targeting the biomechanical properties of tumor cells is a promising and novel strategy for anti-tumor therapy. Our study provides valuable insights into the role of the SPXJ formula in HCC therapy. It represents an innovative treatment approach with significant potential to advance the field of oncology and improve the prognosis of HCC patients (Figure 8).

Figure 8 The effects of Shi-pi-xiao-ji-serum on cellular stiffness. SPXJ: Shi-pi-xiao-ji; MβCD: Methyl-β-cyclodextrin; ACAT1: Acetyl-coA acetyltransferase 1.