The Andrographolide Analogue 3A.1 Synergizes with Taxane Derivatives in Aggressive Metastatic Prostate Cancers by Upregulation of Heat Shock Proteins and Downregulation of MAT2A-Mediated Cell Migration and Invasion.

Conventional treatment with taxanes (docetaxel-DTX or cabazitaxel-CBZ) increases the survival rates of patients with aggressive metastatic castration-resistant prostate cancer (mCRPC); however, most patients acquire resistance to taxanes. The andrographolide analog, 19-tert-butyldiphenylsilyl-8,7-epoxy andrographolide (3A.1), has shown anticancer activity against various cancers. In this study, we investigated the effect of 3A.1 alone and in combination with DTX/CBZ against mCRPC and their mechanism of action. Exposure to 3A.1 alone exhibited a dose- and time-dependent antitumor activity in mCRPC. Chou-Talalay's combination index (CI) values of all 3A.1 + TX combinations were less than 0.5, indicating synergism. Co-treatment of 3A.1 with TX reduced the required dose of DTX and CBZ (P < 0.05). Caspase assay (apoptosis) results concurred with in vitro cytotoxicity data. RNA sequencing (RNAseq), followed by ingenuity pathway analysis (IPA), identified that upregulation of heat-shock proteins (Hsp70, Hsp40, Hsp27, and Hsp90) and downregulation of MAT2A as the key player for 3A.1 response. Furthermore, the top treatment-induced differentially expressed genes (DEGs) belong to DNA damage, cell migration, hypoxia, autophagy (MMP1, MMP9, HIF-1α, Bag-3, H2AX, HMOX1, PSRC1), and cancer progression pathways. Most importantly, top downregulated DEG MAT2A has earlier been shown to be involved in cell migration and invasion. Furthermore, using in silico analysis on the Cancer Genome Atlas (TCGA) database, this study found that MAT2A and highly co-expressed (r > 0.7) genes, TRA2B and SF1, were associated with worse Gleason score and nodal metastasis status in prostate adenocarcinoma patients (PRAD-TCGA). Immunoblotting, comet, and migration assays corroborated these findings. These results suggest that 3A.1 may be useful in increasing the anticancer efficacy of taxanes to treat aggressive PCa. SIGNIFICANCE STATEMENT: The andrographolide analogue, 19-tert-butyldiphenylsilyl-8,7-epoxy andrographolide (3A.1), showed anticancer activity against metastatic castration-resistant and neuroendocrine variant prostate cancers (mCRPC/NEPC). Additionally, 3A.1 exhibited synergistic anticancer effect in combination with standard chemotherapy drugs docetaxel and cabazitaxel in mCRPC/NEPC. Post-treatment gene expression studies revealed that heat shock proteins (Hsp70, Hsp40, Hsp27, and Hsp90) and MAT2A are important in the mechanism of 3A.1 action and drug response. Furthermore, DNA damage, cell migration, hypoxia, and autophagy were crucial pathways for the anticancer activity of 3A.1.

Identification and Characterization of Key Differentially Expressed Genes Associated With Metronomic Dosing of Topotecan in Human Prostate Cancer.

Repetitive, low-dose (metronomic; METRO) drug administration of some anticancer agents can overcome drug resistance and increase drug efficacy in many cancers, but the mechanisms are not understood fully. Previously, we showed that METRO dosing of topotecan (TOPO) is more effective than conventional (CONV) dosing in aggressive human prostate cancer (PCa) cell lines and in mouse tumor xenograft models. To gain mechanistic insights into METRO-TOPO activity, in this study we determined the effect of METRO- and CONV-TOPO treatment in a panel of human PCa cell lines representing castration-sensitive/resistant, androgen receptor (+/-), and those of different ethnicity on cell growth and gene expression. Differentially expressed genes (DEGs) were identified for METRO-TOPO therapy and compared to a PCa patient cohort and The Cancer Genome Atlas (TCGA) database. The top five DEGs were SERPINB5, CDKN1A, TNF, FOS, and ANGPT1. Ingenuity Pathway Analysis predicted several upstream regulators and identified top molecular networks associated with METRO dosing, including tumor suppression, anti-proliferation, angiogenesis, invasion, metastasis, and inflammation. Further, the top DEGs were associated with increase survival of PCa patients (TCGA database), as well as ethnic differences in gene expression patterns in patients and cell lines representing African Americans (AA) and European Americans (EA). Thus, we have identified candidate pharmacogenomic biomarkers and novel pathways associated with METRO-TOPO therapy that will serve as a foundation for further investigation and validation of METRO-TOPO as a novel treatment option for prostate cancers.

Apoptosis Induction and Antimigratory Activity of Andrographolide Analog (3A.1)-Incorporated Self-Assembled Nanoparticles in Cancer Cells.

Andrographolide analog, namely 19-tert-butyldiphenylsilyl-8,17-epoxy andrographolide (or 3A.1) has been reported to be a potential anticancer agent for several types of cancer. Due to its poor aqueous solubility, 3A.1 was incorporated within self-assembly polymeric nanoparticles made of naphthyl-grafted succinyl chitosan (NSC), octyl-grafted succinyl chitosan (OSC), and benzyl-grafted succinyl chitosan (BSC). These 3A.1-loaded nanoparticles were nanosized (< 200 nm) and spherical in shape with a negative surface charge. 3A.1-loaded nanoparticles were produced using a dropping method, which 40% initial drug adding exhibited the highest entrapment efficiency. The release of 3A.1 from the 3A.1-loaded nanoparticles displayed a delayed release pattern. Under acidic conditions (pH 1.2), there was no free drug release. After the pH was adjusted to 6.8, a high cumulative 3A.1 release was obtained which was dependent on the hydrophobic moieties. These 3A.1-loaded pH-sensitive nanoparticles proved to be beneficial for specifically delivering anticancer drugs to the targeted colon cancer sites. In vitro anticancer activity against HT-29 found that the 3A.1-loaded nanoparticles had significantly lower IC50 than that of the free drug and promoted apoptosis. Additionally, in vitro wound-healing migration on HN-22 revealed that free 3A.1 and the 3A.1-loaded nanoparticles inhibited cell motility compared with untreated cells. These pH-sensitive amphiphilic chitosan nanoparticles may be promising nanocarriers for oral anticancer drug delivery to colorectal cancer cells. Graphical abstract ᅟ.