Globally, cancer is currently the second leading cause of death and is expected to be responsible for approximately 9.6 million deaths in 2018 . Owing to the unprecedented understanding of the molecular pathways that drive the development and progression of human cancers, novel targeted therapies have become an exciting new development for anti-cancer medicine. The researches on targeted therapies achieved a great progress that the application of targeted agents tremendously improve the survival time of partial patients with acute myeloid leukemia, breast, gastric, and colon cancers. However, when it comes to some refractory cancers, such as HCC, there is still a hug gap. Hepatocellular carcinoma is the sixth most common malignancy and the fourth leading cause of cancer-related deaths worldwide . In recent years, the first line targeted drugs for HCC are sorafenib and its alternative lenvatinib. However, the anticancer efficacy of sorafenib is unsatisfactory because it only prolongs the overall survival (OS) period by approximately three months compared with placebo. The efficacy of lenvatinib is better than that of sorafenib, but the improvement is limited . Thus, there is an urgent need to identify new potent anticancer drugs for HCC patients.
When it comes to drug screening for drug discovery and development, enormous amounts of money and time must be spent to obtain clinically approved drugs . Even after the long and costly process to identify potential compounds, ~80% of drugs failed during clinical trials and the most common reason for failure was lack of efficacy . Thus, choosing a more effective cancer model that has higher predictive power for anticancer drug screening can greatly improve the success rate of drug discovery and reduce the cost of the screening process. In recent years, the rapid development of 3D culture techniques has provided various pre-clinical models for drug screening. Compared with the traditional drug screening model of flattening (2D) cultured cancer cell lines, 3D cultured cancer models have unique advantages in mimicking the microenvironment and 3D structure of in vitro tumors, and are good at maintaining the functional characteristics of original cells [27, 28]. Therefore, the 3D cultured cancer model has higher predictive power than the 2D model and is better for drug screening.
Spheroid culture is a type of 3D culture technology that has been developed for nearly three decades . This technique is characterized by its low technical difficulty and cost, and it can well imitate the three-dimensional structure and metabolic function of tumors in vivo [30, 31]. Thus, in a previous study, we developed a novel spheroid culture device for anti-tumor drug screening . We used the 3D printing technique to print a resin mold with a fine structure on a micrometer-scale, and then we used the printed mold to shape the agarose substrate in the cell culture well of an ordinary 96-well plate to obtain a special structure, such as a micro-well and liquid exchanging platform. Finally, makes the ameliorated 96-well plate realizing the function of high-throughput 3D culture of cancer cells. Our invented 3D culture device is low cost, reusable, suitable for various cells including primary cancer cells, and is effective in controlling the size and shape of cultured spheroids, meaning that it is a proper tool in 3D drug screening. Therefore, in this study, we designed a set of schemes to screen the anti-HCC drugs by using our homemade 3D culture devise for the aims that find out a potential anti-HCC molecular targeted agent and hope to establish an effective method for in vitro 3D drug screening.
First, we conducted a three-round 3D drug screening on HCC cell lines using our homemade spheroid culture device, and finally screened out CUDC-907 as a potential anti-HCC agent from 19 inhibitors. The results of the drug sensitivity test showed that CUDC-907 has a potent inhibitory effect on HCC cell lines at a low concentration and its efficacy is obviously better than that of the first-line anti-HCC drug sorafenib. Furthermore, functional experiments showed that CUDC-907 inhibited proliferation, caused G2/M arrest, and induced apoptosis in HCC cells. The above evidence suggests that CUDC-907 is a promising anti-HCC agent. CUDC‐907, a dual PI3K/HDAC inhibitor, has been proposed to have therapeutic potential in many hematopoietic malignancies and solid cancers, such as chronic lymphocytic leukemia, acute myeloid leukemia, thyroid cancer, and pancreatic adenocarcinoma [14-17]. It has been recently granted Fast Track designation for the treatment of adults with relapsed or refractory diffuse large B-cell lymphoma by the US Food and Drug Administration . Importantly, it is the first time that our research team found that CUDC-907 has a potent inhibitory effect on HCC cells.
Cancer cell lines are the oldest and most widely used models in cancer research because of their low cost and convenience in manipulation. Due to their ability to grow infinitely, they are well applicable in high-throughput screenings. However, the preparation of cell lines from a tumor results in loss of the 3D in vivo structure and diversity of cell populations; thus, these models only partly represent the origin tumor . Besides, the long-term in vitro culture causes genetic drift and is easily cross-contaminated . These disadvantages significantly limit the predictive power of cancer cell line models in drug screening. Fortunately, the improvement of the culture technique of primary cancer cells helps in these problems. Primary cancer cells maintain the original genetic background and different subgroups of cancer cells; therefore, primary tumor cells are more representative and suitable for drug screening [34-36]. Based on these findings, in this study, we extracted primary HCC cells for the 3D drug-sensitive test. Seven cases of primary cells including six cases of HCC and one case of hepatocyte were successfully extracted and cultured. The results of the drug sensitivity test showed that the inhibitory efficacy of CUDC-907 on different patient-derived primary cells varied. The IC50 of CUDC-907 in different cases of primary HCC ranged from 1.7 to 103 nM (0.86~52.38 ng/mL), which is much lower than the IC50 of sorafenib, and the killing ability of CUDC-907 in primary hepatocyte is weak. These results suggest that the in vitro inhibitory effect of CUDC-907 on primary HCC cells is stronger than sorafenib but has individual difference in drug responses.
Although a previous report pointed out that the peak plasma concentration of CUDC-907 was around 5 ng/mL, the concentration of CUDC-907 in tumor samples could reach approximately 69.5 ng/g . Whether CUDC-907 is effective for HCC treatment in vivo remains unknown. We next conducted an animal study to further prove the effect of CUDC-907 in vivo. Initially, we planned to establish patient-derived xenograft (PDX) mouse models by subcutaneous engraftment of tumor species. However, this experiment failed as the engrafted tissues did not grow in vivo. For substitution, we established an SMMC-7721 HCC xenograft. The results of animal experiments suggested that CUDC-907 could significantly suppress the growth of HCC tumors in vivo. All of these evidences supports the opinion that CUDC-907 might be a potential anti-HCC agent and the outcome of the experiment implies that the 3D in vitro drug screening method we used in this study is a valuable method for drug screening and discovery.
Previous reports have shown that CUDC-907 is a dual-acting inhibitor of PI3K and HDAC, and exhibits predominant anticancer effects in c-Myc-driven tumors [38, 39]. The c-Myc oncoprotein is an essential transcription factor that regulates the expression of many genes involved in cell growth, proliferation, and metabolic pathways . The deregulation and enhancement of c-Myc play an essential role in the carcinogenesis and progression of virous cancer, including HCC . In order to have a profound understanding of the anti-HCC effect of CUDC-907, we investigated the expression level of possible target molecules of CUDC-907. We used western blotting to investigate the impact of CUDC-907 on HDAC, PI3K, and c-Myc in HCC cells. Western blotting of HCC cell lines and xenograft tissues showed that CUDC-907 can inhibit the HDAC and PI3K/AKT/mTOR pathway and suppress the expression of c-MYC. It is worth mentioning that the PI3K/AKT/mTOR pathway is also a central regulator of various oncogenic processes including cell growth, proliferation, metabolism, and angiogenesis in HCC . Thus, the dual inhibition of c-Myc and PI3K/AKT/mTOR may be the main reason that contributes to the potent effect of CUDC-907 on HCC.