Antitumor efficacy and molecular mechanism of lenvatinib combined with vitamin K2 against hepatocellular carcinoma.

Introduction
In China, primary liver cancer ranks fourth in incidence and second in mortality among malignant tumors, posing a serious threat to public health [1]. Primary liver cancer is divided into three types, hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and mixed hepatocellular carcinoma-cholangiocarcinoma (cHCC-CCA), among which HCC accounts for more than 75%. HCC is characterized by insidious onset, high malignancy, rapid progression, and high recurrence rate. Upon diagnosis, most patients are already in the intermediate to advanced stages of the disease and thus miss the opportunity for surgical resection [2]. Consequently, for such patients, the treatment protocols endorsed by both Chinese and international guidelines are systemic anti-cancer therapies, encompassing molecular-targeted treatments and immunotherapies [3]. This approach not only curbs the advancement of tumors but also extends the overall survival rates of patients. In China, the first-line targeted therapies approved for advanced hepatocellular carcinoma (HCC) include sorafenib and lenvatinib.
Sorafenib was the only drug approved for systemic treatment of HCC patients before 2017 and is a tyrosine kinase inhibitor. It can block the activity of vascular endothelial growth factor receptor (vascular endothelial growth factor, VEGF), platelet-derived growth factor receptor and Raf kinase, and can extend the average overall survival of patients with advanced HCC by 2.8 months [4]. Lenvatinib is a potent tyrosine kinase inhibitor that exerts its therapeutic effects by suppressing the activities of VEGF receptors (VEGFRs), fibroblast growth factor receptors (FGFRs) 1 ~ 4, as well as platelet-derived growth factor receptors (PDGFRs), including RET and KIT [5]. Analysis showed that there was no significant difference in the median survival time between the sorafenib treatment group and the lenvatinib treatment group (sorafenib 10.2 months vs lenvatinib 15.0 months), but the disease progression time in the lenvatinib group (9.2 months) and objective response rate (43.8%) were significantly higher than those in the sorafenib group (3.6 months, 13.2% respectively) [6]. Therefore, lenvatinib became an alternative treatment to sorafenib and was approved by the FDA as a first-line treatment for advanced HCC in 2018.
Recent research has revealed that certain vitamins, including Vitamin K2, possess potential preventative and therapeutic benefits against liver cancer [7]. Vitamin K2, a naturally occurring lipophilic vitamin, is primarily utilized for the treatment of osteoporosis and to facilitate the process of blood coagulation [8]. Existing experiments show that vitamin K2 can inhibit the growth of liver cancer cells. An in vitro study conducted in 2021 found that vitamin K2 also inhibited the growth of HSD17B4-induced transplanted tumors [9]. In vitro cell experiments have shown that vitamin K2 can significantly improve the inhibitory effect of sorafenib on the growth of liver cancer cells (HepG2, Hep3B and HuH7). In addition, vitamin K2 (1 μM) combined with low-dose sorafenib (2.5 μM) produces similar in vitro anti-tumor effects as high-dose sorafenib (5 μM) alone [10]. A clinical trial conducted in 2021 demonstrated that the combination of vitamin K with sorafenib significantly extended progression-free survival in patients with liver cancer. This finding underscores the potential synergistic effect of vitamin K in enhancing the efficacy of sorafenib, a standard treatment for advanced hepatocellular carcinoma (HCC) [11]. However, the synergistic potential of vitamin K with other hepatocellular carcinoma drugs remains uncertain.
Lenvatinib has the same target as sorafenib, and its efficacy is better than sorafenib. The synergistic relationship and potential mechanisms of action between lenvatinib and vitamin K2 have not yet been documented in the literature. This study aimed to investigate whether there is a synergistic effect between lenvatinib and vitamin K2 in inhibiting the growth of HCC. Our research team has been dedicated to unraveling the molecular mechanisms underlying the development and treatment of liver cancer. In our comparative experiments involving the human liver cancer cell line HuH-7 and liver fibrosis cells, we discovered a significant upregulation of the SERHL-miR-1269a-BCL2L2 ceRNA pathway. This finding suggests that the pathway could serve as a potential diagnostic biomarker for liver cancer [12]. In this study, we used human liver cancer Huh7 cells as a model to explore the effect of lenvatinib combined with vitamin K2 on the growth and function of liver cancer cells. The mechanism of action underlying the combination therapy was also elucidated, thus providing insights for the development of novel therapeutic strategies against hepatocellular carcinoma.

Methods

Reagents
The human liver cancer cell line Huh7 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Lenvatinib (Selleck, S1164) and vitamin K2 (Selleck, S5082) were purchased from Selleck. The culture medium, fetal bovine serum (FBS), penicillin–streptomycin solution (PS) and 0.25% trypsin solution were purchased from Gibco, USA. Crystal violet stain, chemiluminescence biotin-labeled nucleic acid detection kit, RIPA lysis buffer, BCA protein assay kit (Beyotime Biotechnology) and secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG) were purchased from Shanghai Beyotime Biotechnology Ltd.

Cell culture
Human liver cancer cell HuH-7 was cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100u/mL), L-glutamine (2 mM), and streptomycin (100 μg/mL) at 37° C with 5% CO2 incubator.

Determination of half-maximal inhibitory concentration (IC50)
HuH-7 cells were seeded in a 96-well plate and cultured in an incubator. After 24 h of culture, different concentrations of lenvatinib (0 ~ 32 nM) and vitamin K2 (0 ~ 32 mM) were added respectively. After drug treatment for 120 h, one hundred microliters of CTG reagent were added to each well and mixed thoroughly. Then, 100 µL of the supernatant from each well was transferred to a new microplate, and the absorbance was measured. Finally, a curve was drawn based on absorbance and the IC50 was calculated.

Cell proliferation
Cells in the logarithmic growth phase were treated with drugs for 24 h. The cells treated with lenvatinib, vitamin K2, and lenvatinib combined with vitamin K2 for 24 h were digested with trypsin, centrifuged, and resuspended. The cell concentration was diluted to 1/10 (20 μL cell suspension added to 180 μL PBS). Ten microliters of diluted cells were taken and counted under a microscope to determine the exact cell concentration. Based on the cytotechnical results, dilute the cell concentration with culture medium to 1500 cells/100 μL. Transfer the diluted cell suspension to a 96-well plate, 100 μL per well. CTG was added to the 96-well plate at 100 μL per well. The OD value of the cells added with CTG was measured on a multifunctional microplate reader. 24 h after drug treatment was recorded as Day 0. On Day 0, Day 3 and Day 5, measure the OD value once using the above method. Calculate the cell proliferation rate of each group based on the OD value measured each time.

Cell migration and invasion
After treated with different drugs for 24 h, the cells were digested, centrifuged and resuspended in culture medium. According to the cell counting results, dilute the cell concentration to 2–4 × 104/100 μL. Cell suspension was seeded into the upper chamber of the Transwell chamber at a density of 100 μL/well for migration assay, or into chambers coated with Matrigel for the invasion assay, and complete culture medium (600 μL, containing 10% FBS) was added to the lower chamber. After culturing for 48 h, the cells were fixed with absolute ethanol and stained with 1% crystal violet. Five fields of view were randomly selected and photographed under an inverted microscope. Add 200 μL of 33% acetic acid solution to the 24-well plate. Each chamber was then individually soaked in a well of a 24-well plate for full destaining. Pipette 100 μL of the destained solution from each well of the 24-well plate, and read the absorbance at a wavelength of 560 nm. Three multiple wells were set up in each group, and the relative migration and invasion rates of each group were calculated.

Animal study
This study was approved by Medical Ethics Committee of Changzhou Third People's Hospital (IRB-S0P-07-AF02-2). Female BALB/c nude mice (4–6 weeks old) were purchased from Beijing VitalRiver Laboratory Animal Technology Co., Ltd. and housed under specific pathogen-free (SPF) conditions (temperature 22 ± 2 °C, 50–60% relative humidity, 12 h light/dark cycle) with ad libitum access to food and water. Mice were randomly assigned into four groups (n = 5 per group): Control group (vehicle treatment), Lenvatinib monotherapy group, Vitamin K2 monotherapy group, Combination treatment of lenvatinib and vitamin K2 group. For tumor inoculation, Huh7 cells in the logarithmic growth phase were harvested, washed with PBS, and resuspended at 5 × 10^6 cells/100 μL PBS per mouse. Cell suspensions were injected subcutaneously into the right flank of each mouse using a 27-gauge needle. Tumor size was measured every 2–4 days with a digital caliper, and tumor volume was calculated using the formula: volume = (width2 × length)/2. Drug treatments began when tumor volume reached approximately 100 mm3 (day 7 after inoculation). Lenvatinib was administered at 10 mg/kg/day via oral gavage; vitamin K2 at 30 mg/kg/day via intraperitoneal injection. The combination group received both agents at the same respective doses and schedules. The control group received equivalent volumes of vehicle (0.5% carboxymethylcellulose sodium solution for lenvatinib; saline containing 0.1% ethanol for vitamin K2). All treatments were administered once daily for a total of 30 days. Mice were euthanized 30 days after cell inoculation or if the longest dimension of the tumors reached 2.0 cm. Paraffin-embedded sections were dewaxed and rehydrated, then the samples were treated with proteinase K, fixed in 4% paraformaldehyde. IHC assays were performed using the following primary antibodies: anti-Ki-67.

Western blot
The cells treated with the drug for 24 h were used to extract total protein with RIPA lysis buffer. The protein concentration of the protein solution was determined using a BCA kit. According to the concentration of the protein solution, prepare a protein loading solution with a mass of 40 μg/well. Load the protein loading solution onto 10% SDS-PAGE for electrophoresis, and then transfer the protein in the gel to a PVDF membrane. The above PVDF membrane was blocked in skim milk powder solution (5%) for 1 h. After blocking, add primary antibody solution and incubate overnight at 4 °C. The dilution ratio of each primary antibody is as follows: OSR2 (Abcam, ab129897), 1:1000; ETFBKMT (Invitrogen, PA5-96,594), 1:500; FBXO32 (Proteintech, 67,172–1-Ig), 1:300; ATF5 (Proteintech, 67,066–1-Ig), 1:500; PTPN14 (Abcam, ab138085), 1:1000; GAPDH (Proteintech, 10,494–1-AP), 1:3000. Wash the membrane three times with PBST. Add horseradish peroxidase-labeled goat anti-rabbit/mouse secondary antibody (diluted at a ratio of 1:4000) and incubate at room temperature for 2 h. Develop with ECL chemiluminescent agent. After development, the film is scanned, followed by data acquisition and analysis.

Real-time fluorescence quantitative PCR (RT-qPCR)
Cells were collected, and total RNA was extracted using Trizol method. Using PrimeScript RT Reagent Kit with gDNA Eraser reagent to perform genomic DNA removal and reverse transcription reaction on the extracted RNA. The total amount of reverse transcribed RNA was 1 μg. RT-qPCR was performed using QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher) instrument and 2 × SYBR Green real-time PCR master mix kit. Using GAPDH or 18S as the internal reference gene. Collect data and quantify gene expression using a relative quantification method, the 2-ΔΔCt method.

Transcriptome sequencing
To explore the molecular mechanism of the synergistic antitumor activity of lenvatinib combined with vitamin K2, Huh7 cells treated with lenvatinib, vitamin K2, and the combination of lenvatinib and vitamin K2 were subjected to transcriptome sequencing. Briefly, each group contained three independent biological replicates. Reads were aligned to the human reference genome (GRCh38/hg38) using STAR, quantified with featureCounts, and differential expression analysis was conducted using DESeq2. Genes with p-values < 0.05 were considered significant. This additional detail ensures the transparency and reproducibility of our RNA-seq analysis.

Statistical analysis
All data were processed using SPSS 23.0 statistical software. Measurement data are expressed as mean ± standard deviation (Mean ± SD). The Kruskal–Wallis test was used to compare the means of the four groups. P < 0.05 was considered as a statistically significant difference.

Results

Identification of IC50 of lenvatinib and vitamin K2
In Huh7 cells, cells were treated with different concentrations of lenvatinib and vitamin K2. CTG experiment results show that the IC50 of lenvatinib and vitamin K2 was 10.43 nM and 13.42 mM respectively (Fig. 1A, B).

Lenvatinib combined with vitamin K2 has synergistic anti-tumor effects
Huh7 cells were treated with lenvatinib (10.43 nM), vitamin K2 (13.42 mM), and lenvatinib combined with vitamin K2 respectively. Results showed that compared with control group cells, both lenvatinib and vitamin K2 could significantly inhibit cell proliferation and colony formation. The combined application of the two drugs more significantly inhibited cell proliferation and colony formation, indicating the synergistic anti-tumor effect of lenvatinib and vitamin K2 (Fig. 1C-E).

Lenvatinib combined with vitamin K2 inhibits metastasis and invasion of hepatocellular carcinoma
Huh7 cells were treated with lenvatinib (10.43 nM), vitamin K2 (13.42 mM), and lenvatinib combined with vitamin K2. The results showed that compared with the control group, lenvatinib and vitamin K2 significantly inhibited tumor metastasis and invasion. And the combined treatment of the two showed a more obvious inhibitory effect, (Fig. 2).

Molecular mechanism of synergistic antitumor activity of lenvatinib and vitamin K2
To explore the molecular mechanism of the synergistic antitumor activity of lenvatinib combined with vitamin K2, Huh7 cells treated with lenvatinib, vitamin K2, and the combination of lenvatinib and vitamin K2 were subjected to transcriptome sequencing. The differentially expressed genes were defined as those with a p-value of less than 0.05. The results showed that compared to the control group, treatment with lenvatinib (Fig. S1A), vitamin K2 ((Fig. S1B), and the combination of lenvatinib and vitamin K2 (Fig. S1C) all significantly induced changes in gene expression.
The above differentially expressed genes were subjected to pathway enrichment through Kyoto Encyclopedia of Genes and Genomes (KEGG). The enrichment results show that the lenvatinib group (Fig. S2A), vitamin K2 group (Fig. S2B), and lenvatinib combined with vitamin K2 group (Fig. 3) can all enrich the cell cycle signaling pathway (cell cycle). Correspondingly, Gene Set Enrichment Analysis (GSEA) was performed and similar results were obtained, both of which can significantly enrich the cell cycle (Fig. S3A, 3B; Fig. 4).

Lenvatinib combined with vitamin K2 coordinately regulates cell cycle signaling pathways
In view of the above experimental results, a comprehensive analysis was conducted on the differential genes in the cell cycle generated by the enrichment of lenvatinib, vitamin K2, and lenvatinib combined with vitamin K2. The results showed that there were 5 cell cycle genes that were changed in all groups (Fig. 5). Among them, OSR2, ETFBKMT and FBXO32 were up-regulated (Fig. S4A) and ATF5 and PTPN14 were down-regulated (Fig. S4B). In order to verify the above sequencing data, cells were treated with lenvatinib, vitamin K2, lenvatinib combined with vitamin K2, and the mRNA (Fig. 6) and protein levels (Fig. 7) of OSR2, ETFBKMT, FBXO32, ATF5 and PTPN14 genes were detected. Moreover, functional studies further demonstrated that knockdown of OSR2 or FBXO32 attenuated the synergistic anti-proliferative and pro-apoptotic effects of the combination therapy, whereas overexpression of PTPN14 antagonized such synergistic efficacy, confirming the causal roles of these genes in mediating the combined action of lenvatinib and vitamin K2 (Fig. 8 and Fig. S5A-C).

Lenvatinib combined with vitamin K2 demonstrated a stronger in vivo anti-tumor effect
To further validate the synergistic anti-tumor effects observed in vitro, we conducted an in vivo study using BALB/c nude mice subcutaneously implanted with Huh7 cells. Mice were treated with lenvatinib, vitamin K2, or a combination of both agents. Tumor growth was monitored over 30 days, and the results revealed that the combination therapy significantly inhibited tumor progression compared to monotherapy groups or the control group. The tumor volume in the combination group was markedly reduced compared to the control group, while lenvatinib and vitamin K2 alone showed moderate and modest reductions, respectively (Fig. 9A-C).
Immunohistochemical (IHC) analysis of tumor tissues demonstrated a pronounced decrease in Ki-67 expression (a proliferation marker) in the combination group, further corroborating the enhanced anti-proliferative effects of the dual treatment (Fig. 8D-8E). These results collectively indicate that the combination of lenvatinib and vitamin K2 not only suppresses tumor growth more effectively but also modulates key molecular targets involved in cell proliferation.

Discussion
Hepatocellular carcinoma stands as one of the most prevalent and lethal malignant tumors globally, and China accounts for about 50% of new liver cancer cases and deaths in the world every year [13]. In the complex and evolving realm of treating intermediate and advanced liver cancer, drug therapy has emerged as an indispensable cornerstone, among which targeted drugs have carved out a preeminent position [1]. For patients with unresectable intermediate and advanced stages, neoadjuvant or conversion therapy followed by surgical resection of the tumor may achieve better clinical outcomes [14, 15]. Therefore, lenvatinib monotherapy or lenvatinib-based combination therapy has gradually entered a new stage of conversion, neoadjuvant, and postoperative adjuvant treatment exploration.
Studies have found that vitamin K2 can inhibit the growth of liver cancer cells. Qin Chuanrong et al. [16] found that vitamin K2 induces apoptosis of liver cancer cells by upregulating the expression of BTG2, thereby inhibiting the growth of tumor cells. In vivo experimental literature reports that vitamin K2 can also reduce the recurrence rate of liver cancer after surgery and prolong the survival time of patients [17]. Studies have shown that low-dose sorafenib (1.25 mg/kg) combined with vitamin K2 (2 mg/kg) can significantly reduce the number of metastases and inhibit the growth of metastases [18]. Given the shared molecular targets of sorafenib and lenvatinib, this study was strategically designed to dissect the latent influence of lenvatinib in concert with vitamin K2 on the proliferation, migration, and invasion kinetics of HCC Huh7 cells. Employing Western blotting and RT-qPCR, we endeavored to elucidate the underlying molecular mechanisms.
This study verified the synergistic anti-tumor effect of lenvatinib and vitamin K2 in liver cancer cell lines. Huh7 cells were treated with different concentrations of lenvatinib and vitamin K2. The CTG experiment results show that the IC50 concentration of lenvatinib is 10.43 nM and the IC50 concentration of vitamin K2 is 13.42 mM. Cell proliferation experiment results showed that the combined application of the two more significantly inhibited cell proliferation and colony formation. Cell migration and invasion experiments showed that the combined treatment of the two showed a more obvious inhibitory effect.
In order to explore the anti-tumor molecular mechanism of lenvatinib combined with vitamin K2, this study conducted transcriptome sequencing, differential gene analysis, and pathway enrichment analysis on Huh7 cells treated with lenvatinib, vitamin K2, and lenvatinib combined with vitamin K2. Subsequently, the differential genes in the cell cycle generated by the enrichment analysis were comprehensively analyzed. The results showed that there were 5 genes that changed together in the cell cycle (Fig. 5), among which OSR2, ETFBKMT and FBXO32 were up-regulated (Fig. S4A); ATF5 and PTPN14 were down-regulated. In order to further verify the above genes, the mRNA and protein levels of OSR2, ETFBKMT, FBXO32, ATF5 and PTPN14 genes were detected. The results showed that lenvatinib combined with vitamin K2 could significantly increase the expression of OSR2 and FBXO32, and reduce the protein level of PTPN14.
OSR2 (odd-skipped related 2) is a transcription factor screened by Jiang R in 2001. During mouse development, OSR2 is first expressed in vesicles of the mouse mesonephros at E9.25, and is expressed during the development of the craniofacial region, limbs, and kidneys [19]. Amano et al. discovered through gene chip technology that OSR2 plays an important role in cell differentiation and development [20]. However, the exact role of OSR2 in tumorigenesis is unclear. Shinji Kawai's research found that OSR2 can inhibit the early proliferation and late metastasis of tumors [21]. Daniel Uysal found that high OSR2 mRNA was associated with muscle invasion and was an independent prognostic factor for survival in bladder cancer [22]. Similar to the above studies, the results of this experiment showed that OSR2 mRNA was significantly higher in the lenvatinib combined with vitamin K2 group than in other groups. Furthermore, high expression of OSR2 can more significantly inhibit the proliferation and metastasis of tumor cells, and at the same time improve the prognosis of patients with intermediate and advanced liver cancer.
FBXO32 protein is an important component of SCF ubiquitin protein ligase and participates in a variety of cellular processes, such as signal transduction, cell cycle progression, etc. [23]. Studies [24] have found that FBXO32, as an apoptosis regulatory gene, can be negatively regulated by pro-survival signals. In vivo and in vitro studies [25] have shown that FBXO32 can increase tumor cell apoptosis through the TGF-β/SMAD signaling pathway and can significantly inhibit tumor proliferation and metastasis. This study showed that FBXO32 was significantly increased in the lenvatinib combined with vitamin K2 group, indicating that the combination of the two has a synergistic anti-tumor effect.
PTPN14 belongs to the non-receptor protein tyrosine phosphatase family. In breast cancer, PTPN14 inhibits the activity of oncoprotein YAP1 by reducing its content in the nucleus, thereby attenuating the proliferation and transformation of breast cancer cells [26]. In gastric cancer, PTPN14 promotes the invasion and metastasis of gastric cancer cells by affecting the epithelial-mesenchymal transition process [27]. Gretz et al. found that the expression level of PTPN14 increased during liver metastasis of pancreatic cancer cells, suggesting that PTPN14 plays an important role in the metastasis of pancreatic cancer [28]. Studies [29] have shown that patients with pancreatic cancer with high PTPN14 expression levels have shorter postoperative overall survival time and disease-free survival time. The results of this study showed that the expression level of PTPN14 in the lenvatinib combined with vitamin K2 group was significantly lower than that in other groups. It shows that the combination of the two can better inhibit tumor metastasis.
Taken together the findings from previous studies with our own research, we have discovered that vitamin K can synergistically amplify the therapeutic efficacy of targeted liver cancer drugs. This means that when used in conjunction with vitamin K, targeted medications like lenvatinib and sorafenib can achieve equivalent therapeutic outcomes at lower doses, which may lead to a reduction in treatment-related side effects and an enhancement in the quality of life for patients. Consequently, our research could potentially allow a greater number of liver cancer patients to reap the benefits of drug therapy and contribute to the advancement of current treatment strategies.

Conclusions
In conclusion, our research indicates that the combination of lenvatinib and vitamin K2 may synergistically combat liver cancer through the upregulation of OSR2 and FBXO32, as well as the downregulation of PTPN14. Such exploration is essential for establishing more conclusive evidence supporting the use of lenvatinib in conjunction with vitamin K2 for patients with intermediate and advanced stages of liver cancer.

Supplementary Information