Sorafenib plus gemcitabine shows preliminary evidence of additive inhibition of HepG2 cell proliferation, and shows correlative changes in p-ERK and VEGF expression, and suppresses liver tumor growth in NOD/SCID mice.
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Research Article
* These authors contributed equally
Sorafenib plus gemcitabine shows preliminary evidence of additive inhibition of HepG2 cell proliferation, and shows correlative changes in p-ERK and VEGF expression, and suppresses liver tumor growth in NOD/SCID mice.
Liver cancer remains a leading cause of cancer-related mortality due to drug resistance and limited treatment options. There is an urgent need to explore therapeutic strategies to improve outcomes. This pilot study presents a protocol for assessing the potential effects and underlying mechanisms of the combination of sorafenib and gemcitabine in liver cancer. The liver cancer cell line HepG2 was used, and a non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse xenograft model was established. Key procedural steps are demonstrated including: (1) HepG2 cell culture and drug treatment; (2) MTT assay setup, incubation, and absorbance reading; (3) protein extraction, SDS-PAGE, transfer, and immunoblotting for ERK and p-ERK; (4) subcutaneous xenograft establishment in NOD/SCID mice; (5) tumor measurement and volume calculation; and (6) VEGF immunohistochemistry on tumor sections. Critical steps, troubleshooting tips, and equipment specifications are provided. The combination of sorafenib (3 µmol/L) and gemcitabine (10 µmol/L) achieved an inhibition rate of 71.23% ± 6.76% at 72 h, with a q-value of 1.35, suggesting a synergistic effect at this specific dose pair under the conditions tested. Compared with the control group, the combination treatment was associated with reduced p-ERK expression, decreased tumor volume, and qualitatively lower VEGF immunostaining. These data show a correlative association between reduced p-ERK expression and the observed anti-proliferative effect, without establishing causality. In HepG2 cells and a NOD/SCID mouse xenograft model, the combination of sorafenib and gemcitabine shows preliminary evidence of anti-proliferative potential and is associated with reduced VEGF expression; however, this finding does not constitute direct evidence of anti-angiogenic activity, as microvessel density or functional vascular analyses were not performed. This reproducible protocol provides a useful framework for evaluating drug combinations in HCC models and can be adapted for other targeted therapy-chemotherapy regimens, though further mechanistic and dose-validation studies are required before any clinical implications can be drawn.
Liver cancer is one of the most common and deadliest cancers, and it is also one of the fastest-growing malignant tumors1,2. Although the incidence of liver cancer has been decreasing, the overall burden of liver cancer has continued to increase. The global epidemiological data in 2022 revealed that the age-standardized incidence rate of liver cancer in the Global Cancer Observatory (GLOBOCAN) 2022 online database was 15.0 per 100,000 population, while the age-standardized mortality rate of liver cancer was 26.7 per 100,000 population, making it the second leading cause of cancer mortality3. At present, due to the lack of effective treatment of liver cancer, coupled with its insidious onset, most patients die within one year after diagnosis. Sorafenib is the first multi-target kinase inhibitor approved for clinical use, targeting receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and serine/threonine kinases4,5. Sorafenib, an oral multikinase inhibitor, exerts anti-proliferative and anti-angiogenic effects in liver cancer by targeting multiple growth factor pathways. Historically, sorafenib was the first FDA-approved first-line systemic therapy for patients with advanced or unresectable liver cancer6. However, the current first-line treatment landscape for advanced hepatocellular carcinoma (HCC) has expanded significantly. Recommended options now include the immune checkpoint inhibitor combination atezolizumab plus bevacizumab, lenvatinib, and the dual immunotherapy combination durvalumab plus tremelimumab, among others. Despite these advances, sorafenib remains an important first-line option, particularly in regions where immunotherapy combinations are not readily available or contraindicated. Given its widespread use in liver cancer, drug-related adverse events have been a concern7. In the FDA Adverse Event Reporting System (FAERS) database analysis from 2006 to 2023, the adverse events of sorafenib mainly involved the digestive system, skin, and subcutaneous tissues, as well as non-specific physical discomfort such as infection and injury7. While some patients with advanced liver cancer may benefit from treatment with sorafenib, most patients eventually develop resistance or treatment failure, leading to a poor prognosis8,9,10. Despite its use, sorafenib benefits only about 30% of HCC patients; moreover, responders often develop resistance within 6 months11. The molecular pathways involved in sorafenib resistance include extracellular signal-regulated kinase (ERK)/phosphorylated ERK (p-ERK), protein kinase B (Akt), phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), hypoxia-inducible factor 1α (HIF-1α)/HIF-2α, and autophagy-related pathways, with ERK/p-ERK being specifically highlighted and selected in this research11. Gemcitabine is a cytidine analog that enters the body to produce difluorocytidine diphosphate and difluorocytidine triphosphate, which have different inhibitory effects on DNA synthesis12. In hepatobiliary malignancies, gemcitabine has been evaluated as a component of combination chemotherapy. However, gemcitabine has also been clinically reported to exhibit greater drug resistance, limiting the effectiveness of gemcitabine12,13. Therefore, determining the optimal drug regimen effectively and efficiently is extremely challenging. Recent studies have shown that gemcitabine combined with sorafenib may have enhanced therapeutic efficacy based on a novel tumor stemness biomarker prediction14. However, to our knowledge, no study has examined this combination specifically in liver cancer using HepG2 cells and a non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse xenograft model of hepatocellular carcinoma. Therefore, experimental evidence directly validating the additive effects of sorafenib and gemcitabine in liver cancer remains lacking.
The procedures described in this manuscript are widely used but rarely presented in a detailed, visual, step-by-step format suitable for replication by early-career researchers. We hypothesized that the combination of sorafenib and gemcitabine would additively inhibit liver cancer cell proliferation and tumor growth, and that this effect would be associated with downregulation of the ERK/p-ERK pathway and reduced vascular endothelial growth factor (VEGF)-mediated angiogenesis. This study aims to preliminarily explore the inhibitory effect of sorafenib combined with gemcitabine on the proliferation of the HepG2 liver cancer cell line in vitro and in vivo and its mechanism (Supplementary Figure 1). The novelty of this study lies in three aspects: (1) it is the first to test the sorafenib-gemcitabine combination specifically in liver cancer using the widely used HepG2 cell line and a corresponding NOD/SCID mouse model; (2) it investigates the understudied ERK/p-ERK pathway as a potential mechanism; and (3) it provides direct experimental evidence—rather than computational predictions—for the additive effect of this combination in liver cancer. These features distinguish this work from prior combination therapy reports conducted in other tumor types or using different experimental models, particularly in the context of sorafenib-based regimens.
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Ethical approval was provided by the Zhejiang University-Laboratory Animal Welfare and Ethics Review (Approval No. ZJU20250049).
Animals and grouping
A total of 40 female NOD/SCID mice, 4–5 weeks old, weighing 18–20 g, were purchased from a licensed commercial supplier (License No. SCXK (Shanghai) 2013-0006) and housed in specific pathogen-free (SPF)-grade animal rooms. All animal protocols followed standard guidelines. During the experiment, animals were allowed to eat and drink freely, housed in groups (5 mice/cage) in a well-controlled SPF room, with the room temperature maintained at 22 ± 2 °C, 50%–60% humidity, and a light/dark cycle of 12/12 h. The animals were provided food and water ad libitum, and their body weight was monitored every 2 weeks. When mice showed persistent pain, cachexia, tumor ulcers, the tumor size reached a maximum dimension of 2 cm, or other irreversible health problems, euthanasia was carried out by intraperitoneal injection of 0.45% pentobarbital sodium at ≥150 mg/kg body weight followed by confirmation of cessation of breathing and heartbeat, loss of corneal reflex, or application of a secondary physical method if necessary in accordance with the humane termination criteria approved by the Animal Ethics Committee. Sample size (n = 10 per group) was determined based on a power analysis assuming an effect size of 1.2 (derived from pilot experiments), α = 0.05, and β = 0.20 (80% power), plus 20% attrition allowance. Tumor implantation procedures were performed before treatment allocation (see below for xenograft establishment).
The mice were randomly divided into 4 groups using the random number table method after tumor implantation and confirmation of tumor take: control group, sorafenib group, gemcitabine group, and sorafenib combined with gemcitabine group, with 10 mice (5 mice/cage) in each group, to construct the NOD/SCID mouse transplant tumor model. Because tumor tissues during the modeling process may cause poor general condition or even death in NOD/SCID mice, and considering that statistical analysis generally requires an effective sample size, at least 10 mice were included in each group. Random grouping was carried out by independent technicians and the cages were labeled; blinding was not implemented during drug administration or tumor measurement, which is a significant limitation. The remaining tissue sampling, biochemical, and pathological evaluations were all conducted by blinded laboratory personnel. The data were also analyzed blindly by statisticians who had access only to encoded files until the statistics were completed and the data were unblinded. All animals were included in the final analysis. The intraperitoneal route was selected for both drugs to ensure systemic absorption and consistent bioavailability; once-daily dosing was based on previous pharmacokinetic studies showing sustained plasma concentrations with this regimen. The vehicle control group was intraperitoneally injected with 0.2 mL of sterile normal saline. In the sorafenib group, 0.2 mL of 40 mg/kg sorafenib was intraperitoneally injected. In the gemcitabine group, 0.2 mL of 100 mg/kg gemcitabine was intraperitoneally injected. Combination treatment group: 0.2 mL each of 40 mg/kg sorafenib and 100 mg/kg gemcitabine was administered intraperitoneally. Each group was administered once a day for 20 consecutive days. All animals were included in the final analysis, and no mice were excluded from the study. No mortality was observed in any group during the 20-day treatment period. The schedule and doses were selected based on published studies in hepatobiliary cancer models. It should be noted that daily intraperitoneal administration of gemcitabine may cause toxicity. The length and width of the transplanted tumors were measured daily, and the volume of the transplanted tumors was calculated. After 20 days, the animals were euthanized by cervical dislocation under deep anesthesia, and the transplanted tumor tissues were fixed in formalin. Body weight was measured every 2 days throughout the study. Mice were monitored daily for clinical signs of toxicity, including piloerection, hunched posture, reduced mobility, diarrhea, and lethargy. However, serum biochemistry (e.g., alanine aminotransferase, aspartate aminotransferase, and creatinine) and histopathological examination of major organs were not performed. Therefore, subclinical toxicity cannot be excluded.
Drugs and reagents
Sorafenib was dissolved in 10% dimethyl sulfoxide (DMSO) and stored at -20 °C. Before use, the solution was thawed and diluted to a final DMSO concentration of less than 0.5%, which had no effect on cell growth. Gemcitabine was obtained from a commercial source. Radioimmunoprecipitation assay (RIPA) lysis buffer, thiazolyl blue, primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ERK, p-ERK, and VEGF, secondary antibodies, Dulbecco's Modified Eagle Medium (DMEM) high-glucose medium, fetal bovine serum, and the HepG2 cell line were obtained from commercial sources.
Sorafenib and gemcitabine are cytotoxic agents. All drug preparation and handling procedures should be performed in a designated biosafety cabinet while wearing appropriate personal protective equipment (laboratory coat, nitrile gloves, and safety goggles). DMSO, methanol, xylene, and other organic solvents used in this protocol are hazardous and should be handled in a fume hood to avoid inhalation or skin contact. Material safety data sheets (MSDS) for all reagents should be reviewed prior to use.
Cell Culture
The HepG2 cell line was revived and inoculated into cell culture bottles, containing 10 mL of DMEM high-glucose medium (supplemented with 10% fetal bovine serum, 100 U/mL penicillin-streptomycin, and 4 mmol/L glutamine). The cells were incubated at 37 °C in a humidified incubator containing 5% CO₂. The culture medium was replaced every 2 days, and cells were passaged every 4 days to maintain logarithmic growth. The HepG2 cell line was obtained from a commercial source; however, independent cell-line authentication (e.g., short tandem repeat profiling) and mycoplasma testing were not performed in this study.
MTT method for detecting the proliferation inhibition rate
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to determine the proliferation inhibition rate of sorafenib and gemcitabine on the HepG2 cell line. Cells in the logarithmic growth phase were digested using 0.25% trypsin for 2 min, and the digestion was terminated by adding an equal volume of complete medium. After centrifugation at 1000 × g for 10 min, the cells were resuspended in complete medium. The cell density was adjusted to 1 × 105 cells/mL, and 200 µL of the cell suspension was seeded into each well of a 96-well plate. This seeding density was selected based on preliminary optimization experiments in which cell densities ranging from 5 × 103 to 5 × 104 cells/well were tested. The density of 2 × 104 cells/well resulted in linear absorbance increase over 96 h and maintained confluence below 80% at the endpoint, as confirmed by visual inspection under an inverted microscope. To ensure that MTT measurements remained within the linear range, pilot experiments were performed at each time point with vehicle-treated control wells. Medium was replaced every 48 h to prevent nutrient depletion and accumulation of metabolic byproducts. Confluence was monitored daily using an inverted microscope. At 96 h, control wells reached approximately 75%–80% confluence, and no overt signs of nutrient depletion were observed. After cell attachment, the medium was discarded, and the drug-containing medium was added. A drug-free medium was added to the untreated control group (vehicle control for in vivo experiments). The experiment was divided into an untreated control group, a sorafenib group, a gemcitabine group, and a combination treatment group. The doses of sorafenib were 1.5 µmol/L, 3 µmol/L, 4.5 µmol/L, and 6 µmol/L, and the doses of gemcitabine were 5 µmol/L, 10 µmol/L, 15 µmol/L, and 20 µmol/L. The doses of sorafenib combined with gemcitabine were 1.5 µmol/L + 5 µmol/L, 3 µmol/L + 10 µmol/L, 4.5 µmol/L + 15 µmol/L, and 6 µmol/L + 20 µmol/L. Cells in each group were cultured for 24 h, 48 h, 72 h, and 96 h. After the culture, 20 µL of 5 mg/mL MTT was added to each well, and the cells were incubated in a cell incubator for 4 h. The supernatant was carefully discarded, and then 150 µL of DMSO was added to each well, and the plate was shaken for 10 min to completely dissolve the formazan, and the optical density (OD) value of each well was measured at 490 nm using a multifunctional microplate reader. The experiment was repeated three times, and each group had five replicate wells. Data were presented as mean ± SD of the three biological replicates. The cell growth inhibition rate (%) =(1 - OD experimental group /OD untreated group）× 100%. The interaction between the two drugs was judged by the q-value, which was calculated by the formula: q = EAB / (EA + EB - EA × EB). Here, EA and EB are the inhibition rates of drugs A and B when used alone, and EAB is the inhibition rate when the two drugs are used together. When q is greater than 1.15, it indicates a synergistic effect between the two drugs; 0.85–1.15 indicates an additive effect; and q less than 0.85 indicates an antagonistic effect. The q-value method is a simple screening tool and does not substitute for more rigorous synergy analyses. Only a few fixed-ratio dose pairs were tested, which limits the robustness of the synergy claim. Therefore, the term "synergy" in this study refers to q > 1.15 at specific dose pairs and time points, not to a validated additive interaction across the full dose-response matrix.
Western blot analysis of ERK and p-ERK protein expression
Cell culture and experimental grouping were performed as described above. For the detection of ERK and p-ERK by Western Blotting, the dose of sorafenib was 3 µmol/L; the dose of gemcitabine was 10 µmol/L; and the combined dose was 3 µmol/L + 10 µmol/L. After 72 h of treatment, cells were lysed with RIPA lysis buffer, and total protein was extracted from each experimental group. The protein concentration was determined by the bicinchoninic acid (BCA) method. Equal amounts of protein (30 µg per lane) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V for 90 min. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (0.45 µm pore size) at 300 mA for 90 min at 4 °C. After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4 °C with gentle shaking. After three washes with TBST (10 min each), membranes were incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) substrate and imaged using a digital gel imaging system. Exposure times were optimized for each antibody to avoid saturation. Densitometric quantification was performed using ImageJ software using the gel analysis tool. All target protein band intensities were normalized to GAPDH as a loading control, and p-ERK expression was further normalized to total ERK expression. No additional blocking steps beyond 5% non-fat milk were performed. Quantification results were presented as fold change relative to the control group, which was set to 1.0. Each experiment was repeated three times independently. Representative immunoblots are shown.
Immunohistochemical detection of VEGF expression in HepG2 xenograft tumors in NOD/SCID mice
For xenograft establishment: HepG2 cells in the logarithmic growth phase were harvested by trypsinization, washed twice with sterile phosphate-buffered saline (PBS), and resuspended in serum-free DMEM. A total of 2 × 106 cells in 0.2 mL of serum-free DMEM (without Matrigel) were injected subcutaneously into the right axillary region (a rich blood supply area) of each NOD/SCID mouse using a 1 mL syringe to establish a HepG2 subcutaneous xenograft model in NOD/SCID mice. Tumor take was monitored every 2 days. The size of the tumor was measured every two days, and when the tumor diameter reached 0.3–0.5 cm (approximately 7–10 days post-injection, defined as the treatment initiation point), mice were randomly divided into 4 groups for the experiment. The length (L) and width (W) of the subcutaneous xenograft were measured every day using digital calipers, and the tumor volume (V) was calculated according to the formula: V = (L × W2) / 2. All measurements were performed by an investigator blinded to group allocation. After 20 days, the animals were euthanized by cervical dislocation under deep anesthesia, and the xenograft tissue was fixed in 10% neutral buffered formalin for 24 h at room temperature, then paraffin-embedded. Tissue sections (5 µm thick) were cut using a rotary microtome and mounted on poly-L-lysine-coated glass slides. For immunohistochemistry, sections were deparaffinized in xylene (three changes, 5 min each) and rehydrated through a graded ethanol series (100%, 95%, 70%, 50%; 3 min each). Antigen retrieval was performed by heating sections in 10 mM sodium citrate buffer in a microwave for 15 min, followed by cooling at room temperature for 20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min at room temperature. Non-specific binding was blocked with 5% normal goat serum in PBS for 1 h at room temperature. Sections were then incubated with primary anti-VEGF antibody (dilution 1 : 200) overnight at 4 °C in a humidified chamber. After washing with PBS (three times, 5 min each), sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1 : 500) for 1 h at room temperature. Signal was detected using 3,3′-diaminobenzidine (DAB) substrate. Sections were counterstained with hematoxylin for 30 s, dehydrated, cleared, and mounted with coverslips using permanent mounting medium. Imaging was performed using a fluorescence inverted microscope with brightfield settings at ×100 magnification. Representative images are shown for qualitative illustration.
All contaminated tips, tubes, and cell culture waste were collected as biohazardous waste and autoclaved prior to disposal. Chemical waste, including drug-containing solutions, DMSO, xylene, and methanol, was collected in segregated, labeled waste containers and disposed of in accordance with local institutional regulations for hazardous chemical waste.
Statistical analysis
SPSS 19.0 software was used for statistical analysis. Experimental data were expressed as mean ± SD from three independent biological replicates for in vitro experiments, and as mean ± standard deviation (SD) from n = 10 mice per group for in vivo experiments. For comparisons between two groups, two-tailed unpaired t-tests were used. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used. p < 0.05, p < 0.01, and p < 0.001 were reported.
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The inhibitory effect of sorafenib and gemcitabine on HepG2 cell proliferation
Sorafenib and gemcitabine dose-dependently inhibited HepG2 cell proliferation, as shown in Table 1. The combination therapy showed significantly higher inhibition rates at all time points compared to monotherapy. MTT measures metabolic activity, which correlates with cell number but does not directly measure proliferation or cytotoxicity. Therefore, the term "inhibition of prolifera...
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Liver cancer has a high mortality rate due to its insidious onset, rapid progression, and few effective treatments3,15. Advanced liver cancer often exhibits chemotherapy and radiation resistance, and current options, including surgical resection and liver transplantation, remain insufficient15,16 The combination of two or more chemotherapy drugs with different mechanisms of action has been well developed ...
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The authors have nothing to disclose.
The authors have no acknowledgments.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Digital gel imaging system | Bio-Rad Laboratories, Inc., USA | 1708280 | |
| DMEM high-glucose medium | HyClone, USA | SH30243.01 | |
| ERK rabbit monoclonal antibody | Santa Cruz, USA | sc-94 | |
| Fetal bovine serum | Zhejiang Tianhang Biotechnology Co., Ltd., China | 11011-8611/11011-8615 | |
| Fluorescence inverted microscope | Olympus, Japan | IX73 | |
| GAPDH rabbit polyclonal antibody | Santa Cruz, USA | sc-25778 | |
| Gemcitabine | Eli Lilly and Company | 95058-81-4 | |
| Goat anti-rabbit second antibody | Shanghai Shenggong Biological Engineering Co., Ltd., China | D111018 | |
| ImageJ software | National Institutes of Health, Bethesda, MD, USA | Version 1.8.0_112 | |
| Microplate reader | Thermo Fisher Scientific Inc., USA | MK3 | |
| p-ERK rabbit monoclonal antibody | Santa Cruz, USA | 9106 | |
| Refrigerated centrifuge | Changsha Xiangyi Centrifuge Co., Ltd. | DL-5M | |
| RIPA lysis buffer | Biyuntian Biological Technology Research Institute, China | P0013B | |
| Rotary microtome | Leica, Germany | CM3050 | |
| Sorafenib | Bayer Company, Germany | 284461-73-0 | |
| Thiazolyl Blue (MTT) | Sigma, USA | M5655 | |
| Three-gas incubator | Thermo Fisher Scientific Inc., USA | 51026402 | |
| VEGF rabbit monoclonal antibody | BioLegend, USA | B199116 |
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