Colorectal cancer is one of the most common malignant tumors worldwide. Irinotecan (CPT-11), a classic chemotherapeutic agent, plays an important role in its clinical treatment [31]. However, the serious side effects caused by CPT-11 limit its applications. In the last three decades, many studies have reported the synthesis of CPT-11 derivatives to enhance cytotoxicity or minimize adverse events [16, 32, 33]. Unfortunately, no new analogs have been approved so far. Recently, we synthesized a novel CPT-11 derivative, ZBH-01, that showed a higher inhibitory effect on the development of colon tumors, both in vivo and in vitro. In the present study, we explored its molecular mechanisms and demonstrated that ZBH-01 has superior anti-tumor characteristics compared to CPT-11.
DNA topoisomerases, which can be divided into two categories: TOP1 and TOP2, are a class of enzymes that exist in the nucleus. They can catalyze the break and combination of DNA strands, thereby controlling the topological state of DNA. In abnormally proliferating tumor cells, TOP1 and TOP2 are highly expressed. Therefore, topoisomerase inhibitors are an important class of anti-tumor drugs [34]. CPT-11 is a TOP1 inhibitor that disturbs the catalytic cycle of TOP1 by stabilizing the reversible covalent enzyme-DNA cleavable complex. By forming a drug-enzyme-DNA ternary complex during DNA synthesis, CPT-11 triggers the formation of irreversible single-stranded DNA break [35]. Here, a natural amino acid glycine group was used to replace the 4-piperidinopiperidine group to overcome the metabolism drawback of CPT-11. By conjugating the amino group of glycine to the 10-position of SN-38 via a carbamate bond, ZBH-01 was synthesized with the carboxyl group converted to sodium salt to improve water solubility. Surprisingly, ZBH-01 showed more potent antitumor activity in vitro and was rapidly converted to the active SN-38 in both non-enzymatic physiological buffer (pH 7.4) and plasma. The AChE inhibition activity of ZBH-01 was also very lower than CPT-11. Nevertheless, the solubility of ZBH-01 is higher than SN38, about 25.6 mg/mL (sodium salt of ZBH-01) in deionized water, facilitating its absorption. Moreover, the bioconversion of ZBH-01 to the active SN-38 is higher than CPT-11 both in non-enzymatic physiological buffer (pH 7.4) and plasma, which might also contribute to its antitumor potency.
Furthermore, we compared the inhibitory effects of ZBH-01 and CPT-11/SN38 on TOP1 in colon cancer cells. The DNA relaxation assay unexpectedly showed that the inhibition of ZBH-01 on TOP1 was significantly lower than CPT-11 and SN38. This was inconsistent with our previous reports [9, 11]. Then, we confirmed this result using NGS and qRT-PCR. However, the Western blot showed that TOP1 protein was slightly downregulated by ZBH-01, suggesting that ZBH-01 might inhibit TOP1 after transcription. Moreover, we observed that the levels of TOP2A mRNA were significantly repressed in the ZBH-01 group by NGS and qRT-PCR. Hence, we hypothesized that this might be related to the chemical structure of ZBH-01 but we did not explore this problem that requires more accurate experimental designs. We intend to explore these issues in the future.
High-throughput-based gene expression profiling enables the characterization of the relative drug sensitivity of cancer cells [36] and the identification of new drug targets [19]. Thus, to study the ZBH-01 mechanisms, we performed NGS to compare the mRNA expression profiles in colon cancer cells treated with ZBH-01, CPT-11, and SN38. The results showed that ZBH-01 treatment remarkably induced a unique abnormal expression of 1769 DEmRNAs (842 downregulated and 927 upregulated mRNAs) in LS174T cells. The GO and KEGG analysis showed that these DEmRNAs were mainly enriched in DNA replication, p53 signaling pathway, and cell cycle. Then, we filtered one prominent module of the PPI network, consisting of 73 genes mostly associated with the cell cycle. We found that TOP2A was in the center of the module, demonstrating its importance again.
From the above module, we verified the expression of the genes involved in the cell cycle by qRT-PCR. We also verified the expression of other key genes involved in apoptosis, such as BAX, BCL2L1, BID, BIRC3, and BIRC5. Except for a few genes, the expression trend was consistent between NGS and qRT-PCR. There are many reasons for this inconsistency, such as the inaccurate conduction of the experiment, design of primers, and low sample and experimental repetitions.
Then, we performed cell cycle and apoptosis analyses to explore the antitumor activity of ZBH-01. Under the same experimental conditions (50 nmol/L, 24 h), ZBH-01 significantly induced more apoptosis and cell cycle arrest in the G1 phase in LS174T and SW1116 colon cancer cells; while CPT-11 and SN38 mainly induced cell cycle arrest in the S phase. However, they did not induce apoptosis even 48 and 72 h after treatment. At 12 and 18 h, the results showed a similar trend, with ZBH-01 presenting a stronger effect on inducing tumor cell apoptosis. These results were further verified by the MMP and Western blot assays and were consistent with other reports [18].
The members of the BCL2 family are implicated in the intrinsic apoptotic pathway. These proteins can be either pro-apoptotic (BAX and BBC3) or anti-apoptotic (BCL2, BCL2L1, and MCL1) [37]. In our present study, the levels of BAX protein significantly increased, while Bcl-xL decreased after ZBH-01 exposure. Accordingly, the levels of active caspase 3 and cleaved-PARP increased after ZBH-01 treatment. The tumor suppressor P53 is a key regulator in various signaling pathways including DNA damage, cell cycle, and apoptosis [38]. Here, p53 was upregulated after CPT-11, SN38, and ZBH-01 treatments. Among them, ZBH-01 had the strongest effect. These results were consistent with other studies that reported increased expression of p53 after CPT treatment leading to cell cycle arrest [39], and CPT-11 inducing cell cycle arrest in the S- and G2/M-phases [40].
Previous studies have shown that the transcription factor MYBL2 can activate CDC2 and cyclin D1 after being phosphorylated by cyclin A/CDK2. The interaction between these genes plays a decisive role in regulating the transition from the G1 to the S phase. All these genes were detected in our filtered module. Therefore, we verified their levels by qRT-PCR and Western blot. The results showed that the protein levels of CCNA2, CDK2, and MYBL2 were more repressed by ZBH-01 in colon cancer cells compared to treatment with CPT-11 and SN38. This might preliminarily explain our cell cycle experimental results in which ZBH-01 induced cell cycle arrest in the G1 phase (Fig. 11).
Finally, ZBH-01 presented antitumor activity in vivo and comparable tumor inhibitory potential and lower toxicity to CPT-11. These results demonstrated that ZBH-01 is worthy of further studies.
Our study also has some limitations. First, we should analyze more tumor cell lines and other tumor models in mice. Second, we only verified the levels of some DEmRNAs by qRT-PCR. Neither the expressions of gene-encoded proteins nor the gain of or loss of function was evaluated. Furthermore, we did not add corresponding agonists or antagonists to help analyze the mechanisms of ZBH-01. Additionally, there are actual differences between the expression patterns of DEmRNAs in colon cancer tissues and cell lines after drug treatments, and how to fully elucidate these differences might be considered in the future.
In summary, we demonstrated that ZBH-01, a new CPT-11 analog, has a broad spectrum of antiproliferative activity in various human tumor cell lines. By inducing cell cycle arrest and promoting apoptosis, ZBH-01 presented superior antitumor efficacy compared to both CPT-11 and SN38. Finally, ZBH-01 also showed higher tolerability than CPT-11 in vivo.