Anlotinib induces hepatocellular carcinoma apoptosis and inhibits proliferation via Erk and Akt pathway

Abstract

Although anlotinib, a multi-targeted receptor tyrosine kinase inhibitor has been reported have antitumor effects in many preclinical and clinical trials, little is known about its effect on hepatocellular carcinoma (HCC). Here, we have shown the antitumor effects of anlotinib on HCC. Data indicated that anlotinib application significantly inhibited HCC cell viability, proliferation, colony formation, and prompted apoptosis in vitro. Furthermore, animal experiments also illustrated that anlotinib alleviated HCC pro- gression. Mechanically, we demonstrated that anlotinib treatment downregulated the anti-apoptotic protein Bcl-2 and Survivin, but upregulated pro-apoptotic molecule Bax, which accounts for its thera- peutic effect on HCC. Pathway analysis has shown decreased phosphorylation levels of Erk and Akt. Together, this study suggests that anlotinib may have a direct antitumor progression effect on HCC by inhibiting Bcl-2 and Survivin expression, promoting Bax expression via inactivating Erk and Akt path- ways and could be a promising agent treating HCC.

1. Introduction

Hepatocellular carcinoma (HCC) is a highly malignant tumor around the word, and it is also the fifth most prevalent cancer, constituting the second large portion of tumor-related mortality and the fastest rising cancer worldwide [1]. Not like any other cancers, there are some reported risk factors for HCC genesis, such as chronic hepatitis virus (HBV and HCV) infections, alcohol, he- patic adipose infiltration, hepatitis, autoimmune, or chronic cholestatic diseases [2]. In the present, the therapeutic strategies for HCC is surgical resection, conventional chemotherapy, radio- therapy, or liver transplantation [3]. Although great advance has been made in the therapeutic regimen of HCC, the long-term overall survival time of HCC patients remains unchangeable [4], which is mainly because of the late diagnosis and the deficiency of any other non-surgical treatments. In this regard, a better under- standing of the underlying mechanisms of HCC progression is ur- gently needed to search for a new effective agent against HCC.

Anlotinib is a novel oral multi-targeted receptor tyrosine kinase inhibitor that is synthetized to primarily inhibit a group of newly identified kinase, such as VEGFR2/3, FGFR1-4, PDGFR a/b, c-Kit, Ret, Aurora-B, c-FMS, and discoidin domain receptor 1(DDR1), which plays an important role in tumor progression, apoptosis, prolifer- ation and angiogenesis [5e8]. As VEGFR2 is believed to be the major mediator of the VEGF-induced anti-angiogenesis and anti- tumor signaling [9e11], and the IC50 of anlotinib to VEGFR2 is the lowest after measuring anlotinib against a panel of tyrosine kinases including VEGFR1, VEGFR3, PDGFRb, c-Kit, etc. [12]. Additionally, it is reported that anlotinib highly selectively inhibit VEGFR2 [12], we reasonablely hypothesis that anlotinib may exert its efficacy on HCC mainly through inhibits VEGFR2. After being stimulated by VEGF molecules, the intracellular TK domains of VEGFR2 dimerized and autophosphorylated leading to the simultaneous activation of downstream signaling pathways, including the Raf/Mek/Erk1/2 and the PI3K/Akt/mTOR pathway [13e15].

Although anlotinib has shown a promising antitumor activity against diverse malignant tumor types in several preclinical and clinical trials [16e21], no research has estimated its underlying mechanisms in HCC. Here, we are firstly attempt to explore the therapeutic effect of anlotinib on HCC and its possible mechanisms. Our work will provide promising evidence and rationale for clinical therapeutic application of anlotinib in human HCC treatment.

2. Results

2.1. Higher expression of VEGFA is related with HCC and poorer survival time

As illustrated above, anlotinib maybe exert its efficacy via blocking the activation of VEGFR, since VEGFA is the main and the most important component of VEGF family and usually referred to VEGF [22], we assessed VEGFA expression level in HCC and normal tissue, as well as the expression profile in different stages of HCC in public database K-M Plotter [23] and UALCAN [24]. Data indicated that VEGFA expressed higher in HCC compared with the normal tissue (Fig. 1A), to be highlighted, VEGFA expressed higher with the progress of the HCC grade (Fig. 1B). Moreover, we further analysis the relevance between the expression of VEGFA and patients’ sur- vival time, the results indicated that higher VEGFA expression predicted poorer prognosis and shorter survival time (Fig. 1C, 1D and 1E). Together, these findings suggested VEGFA expressed higher in HCC compared with normal tissue, and with the pro- gression of HCC grades, VEGFA expressed higher, additionally, higher VEGFA predicted the poorer prognosis and shorter survival time.

2.2. Anlotinib inhibits HCC progression ex vitro and ex vivo

To test the efficacy of anlotinib on HCC, we co-cultured different concentrations of anlotinib with HCC cells ex vitro and established the xenograft mouse model ex vivo. It is indicated that anlotinib reduced the viability of HCC cells in a concentration-dependent manner by CCK-8 (Fig. 2A) and colony formation assay (Fig. 2B), in Fig. 2C, imagines taken by phase contrast microphotograph also showed poor viability of HCC cells when treated with anlotinib. Furthermore, we also generated the xenograft mouse model to confirm the curative effect of anlotinib in vivo. Two groups of 6e8 weeks old athymic nude mice were subcutaneously injected with 2 106 Huh7 cells. After one week when the subcutaneous tumor mass formed, anlotinib (5 mg/kg) and DMSO 1% were given to the experimental group (n 6) and the control group (n 6) through oral gavage every day. As shown in Fig. 2D, dramatic tumor growth inhibition was observed in the experimental group compared with the control one. These observations were also strengthened by the lower tumor weight and smaller tumor volume in the anlotinib group compared with the control group measured after mice sacrificed (Fig. 2E). In short, our results indicate that anlotinib inhibits HCC progression in vitro and in vivo.

2.3. Anlotinib promotes apoptosis and inhibits proliferation of HCC

Then we further tested the effect of anlotinib on cell apoptosis and proliferation using flow cytometry. Annexin V/7-AAD double staining revealed that anlotinib largely increased the apoptotic percentage of HCC cells (Fig. 3A). In addition, anlotinib treatment could also result in cell cycle arrest in HCC cells (Fig. 3B). Taken together, these data suggested that anlotinib promoted apoptosis and inhibited proliferation of HCC in a concentration dependent manner.

2.4. Anlotinib exerts its efficacy via downregulating Bcl-2 and Survivin and upregulating Bax by inhibiting Erk and Akt pathways

Due to the significant therapeutic efficacy that anlotinib showed in vitro and in vivo, we further tried to find out the underlying mechanism. PCR and Western blot analysis revealed that the expression of anti-apoptotic proteins, including Bcl-2 and Survivin decreased while pro-apoptotic protein Bax increased, thus leading to the decrease in the ratio of Bcl-2/Bax (Fig. 4A and 4B). Mean- while, as Ras/Erk and PI3K/Akt pathways are the main downstream pathways of VEGF signaling, we first detected the main proteins of these two pathways. It turned out that anlotinib downregulated the phosphorylation of Erk and Akt, but there were no significant changes in the total protein levels of Erk and Akt (Fig. 4B). Collec- tively, these observations indicated that anlotinib might inhibit HCC progression by suppressing the expression of Bcl-2 and Sur- vivin, upregulating Bax via inhibiting Erk and Akt pathways (Fig. 4C).

3. Discussion

HCC is one of the top three most death-caused cancers, and the survival and prognosis of HCC patients stays moderate because of delayed diagnosis and the lack of effective treatment strategies. No need to say, there is an urgent clinical demand for an efficient treatment with low toxicity to prolong the overall survival time of patients with HCC.
It is widely known that HCC is less sensitive to most chemo- therapeutic agents than any other cancers [25]. Moreover, most patients have already been at an advanced stage when diagnosed with HCC [26], [27]. The prognosis in these advanced HCC patients are limited. Under this circumstance, targeted therapy shows an attractive prospect in HCC treatment regimens. In 2007, the tyro- sine kinase inhibitor (TKI) sorafenib was approved by FDA for use in advanced HCC as the first-line treatment [11] [28], which means anlotinib has the same potential in the therapy of HCC, moreover, the IC50 of anlotinib to VEGFR1/2/b and FGFR1 is much lower than sorafenib (about 1/4e1/25) [20], which indicated anlotinib may have a better effect to some extent.
Anlotinib as a multi-kinase inhibitor, has shown highly potent and specific inhibition effect against VEGFR2, leading to a signifi- cant pro-apoptosis and anti-proliferation activity against HCC. Be- sides, Phase II trials indicated that anlotinib as a third-line treatment provided significant progression-free survival benefits to patients with refractory advanced non-small-cell lung cancer when compared with placebo, and the toxicity profiles showed good tolerance [24]. Additionally, Phase III clinical trials of anlotinib have been completed in China, and phase II trials against a variety of malignancies are currently underway in the USA [20].

As a mulititarget agent, anlotinib may exert its effect by any other pathways or molecules, which remains further investigation. Although anlotinib is a promising agent, before applying it to HCC patients clinically, more in vivo studies are still needed to do to verify its side effects and adverse events, only figure these out can anlotinib be used safely and extensively.

In summary, we demonstrated that anlotinib inhibited the proliferation and induced the apoptosis of HCC cells possibly by inhibiting the activation of VEGFR2 and the expression of Bcl-2 and Survivin, as well as upregulating Bax expression via inhibiting Erk and Akt signaling transduction pathways. All together, these studies illustrate anlotinib as a promising agent in the treatment of HCC and provide a mechanism evidence for the ongoing clinical investigation of anlotinib as a novel antitumor agent.

Fig. 1. Higher expression of VEGFA is related with HCC and poorer survival time. (A) VEGFA expression in HCC and normal tissue based on UALCAN. (B) VEGFA expression in different grades of HCC and normal tissue based on UALCAN. Kaplan-Meier plot of survival time in VEGFA high and low patients based on database K-M Plotter (D) and UALCAN (C, E). *P < 0.05, **P < 0.01.

Fig. 2. Anlotinib inhibits HCC progression ex vitro and ex vivo. Cell viability with or without anlotinib treatment were measured by CCK-8 (A) and colony formation assay (B). (C) Phase contrast microphotograph images of HCC cells with or without anlotinib for 48 h. (D) Gross image of representative tumors excised after mice sacrificed. (E) Tumor weight and volume were measured at the indicated time. *P < 0.05, **P < 0.01. Scale bar ¼ 100 mm.

Fig. 3. Anlotinib promotes apoptosis and inhibits proliferation of HCC. Cell apoptosis (A) and proliferation (B) with or without anlotinib were measured by flow cytometry. *P < 0.05, **P < 0.01.

4. Methods

4.1. Materials

Anlotinib was kindly given as a gift by Chia Tai Tianqing Co., Ltd (Nanjing, China). Antibodies to Survivin, Bax, Bcl-2, Erk, p-Erk, Akt, p-Akt and GAPDH were purchased from CST (Beverly, MA, USA).

4.2. Database

To uncover the relevance between VEGFA expression and patient survival, we searched the public database K-M Plotter [23] and UALCAN [24].

4.3. Cell culture

Human hepatocellular carcinoma cell HepG2 and Huh7 were purchased from ATCC. Cells were grown in DMEM (HyClone) with 10% FBS (Gibco) and antibiotics (Penicillin 100 U/ml, Streptomycin 100 mg/ml) in 37 ◦C humidified atmosphere with 5% CO2.

4.4. Cell viability assay

The assay was performed as depicted [23]. Briefly, 5000 cells per well were added into 6 well plate and incubated with or without agents for 72 h, then added CCK-8 at 1:10 dilution and cultured for 2 h. OD450 was measured and recorded.

4.5. PCR

The assay was performed as reported [23]. RT-PCR was per- formed utilizing the TaKaRa RT-PCR kit (Japan) in line with the manufacturer’s protocols. Relative quantification (RQ) was calculated by the equation RQ 2—DDCt. The sequences of the primers are listed in Table S1.

4.6. Western blot

The assay was performed as reported [23]. In brief, protein lysate was separated by 10% SDSePAGE electrophoresis and then transferred to a PVDF membrane, next incubated membranes with specific primary and secondary antibodies.

4.7. Colony formation assay

6-well plate contains 1000 cells per well, incubating with the indicated treatment. After 7 days, colonies were fixed and stained with 0.1% crystal violet, colonies were counted manually.

4.8. Flow cytometry analysis

For apoptosis analysis, incubating cells with or without anlotinib for 48 h, afterward cells were harvested and stained by 7-AAD (BD Biosciences) and Annexin-V (BD Biosciences). As for proliferation assay, after treating cells with or without anlotinib for 24 h, do the following assay using EdU Cell Proliferation Kit (Beyotime) as instructed. Briefly, incubating cells with 10 mM Edu for 2 h, then after washing, immobilizing and permeabilizing, cells were treated with Click Additive Solution for 30min and stained with 7-AAD.

4.9. Xenograft mouse model

Mice were maintained in SPF condition at Shanghai No.9 Peo- ple’s Hospital Animal Experimental Center. All animal experiments were approved by the Ethic Committee. For HCC xenograft model, 12 BALB/c athymic nude mice (6e8 weeks old) were subcutane- ously injected with 2 106 Huh7 cells. After one week when the subcutaneous tumor mass formed, anlotinib (5 mg/kg) and DMSO 1% were given to the experimental group (n 6) and the control group (n 6) through oral gavage every day. Tumors were measured by calipers every 7 days and calculated using the formula as 0.5*length*width2. Tumor weight was measured after mice were sacrificed at Day 21.

4.10. Statistics analysis

All experiments were repeated 3 times as indicated. Mean, Standard Error of Mean (SEM), and P values based on t-test were calculated by Excel (Microsoft). Differences were considered sig- nificant at P < 0.05.