Targeting 3-phosphoinositide-dependent protein kinase 1 associated with drug-resistant renal cell carcinoma using new oridonin analogs

Abstract

The current agents used for renal cell carcinoma (RCC) only exhibit the moderate response rate among patients. Development of drug resistance eventually fuels the need of either more potent drugs or new drugs to target the resistant pathways. Oridonin is a diterpenoid isolated from the Chinese medicinal herb Rabdosia rubescens and has been shown to have antitumor activities in many cancers. We previously developed new synthetic methodologies to modify structurally diversified diterpenoids and designed a series of nitrogen-enriched oridonin analogs. In this study, we screened a variety of oridonin analogs based on their cytotoxicity using MTT assay and identify the most potent candidate, namely, CYD-6-17. CYD-6-17 exhibited a high potency to inhibit the in vitro growth of several drug-resistant RCC cells as well as endothelial cells stimulated by tumor cells at nanomolar range. Delivery of CYD-6-17 significantly inhibited RCC tumor growth using xenograft model. Mechanistically, it targeted the 3-phosphoinositide-dependent protein kinase 1 gene that appeared to be a potent regulator of AKT and was associated with patient survival after targeted therapies. This offers a new rational therapeutic regimen of CYD-6-17 to drug-resistant RCC based on its novel mechanism of action.

Figure 1

The effect of oridonin analogs on the growth of RCC cells. (a) The effect of oridonin analogs on the cell growth of HK-2 KD and 786-0 KD determined by MTT assay. The growth inhibition rate was calculated based on the control. (b) Chemical structure of oridonin and CYD-6-17. (c) The effect of CYD-6-17 on the growth of a variety of RCC cell lines determined by MTT assay. The growth inhibition rate was calculated based on the control. (d) The effect of CYD-6-17 on the growth of HUVEC alone or co-cultured with 786-0 KD cells determined by crystal violet assay with OD 555 nm. *P<0.05

Figure 2

The induction of apoptosis and cell cycle arrest RCC cells by CYD-6-17. (a and b) The cell apoptosis of Sor001 and 769-P cells were determined 24 h after the indicated concentration of CYD-6-17 treatment using PI/Annexin V assay (left panel) and the quantification of cell apoptosis as depicted (right panel). (c) Western blot analyses of the expression of apoptotic markers (cleaved PAPR) in Sor001 and 769-P cells after CYD-6-17 treatment. (d) Western blot analyses of the expression of cell cycle regulators in RCC cells after CYD-6-17 treatment. Actin was used as loading control

Figure 3

The mechanism of CYD-6-17 in inhibiting Wnt pathway. (a) 786-0 KD and HK-2 KD cells were treated with control (Con) or CYD-6-17 for 24 h, the total RNA were subjected to Affymetrix GeneChip cDNA array and heat map were depicted for the relative gene expression (fold changes) normalized with Con. Asterisks indicated Wnt-related effectors. (b) Western blot analyses of the expression of GSK3β and β-catenin in RCC cells treated with CYD-6-17. (c) The effect of CYD-6-17 on β-catenin-mediated gene transcriptional activity in RCC cells transfected with TOP luciferase reporter gene. After normalizing with Renilla luciferase activity, the relative reporter gene activity in each cell was calculated based on the control. (d) Determination of cell cycle regulatory factors in Sor001 cells transfected with vector control, WT, or CA β-catenin expression vector for 24 h (left panel) and then treated with CYD-6-17 for 48 h to determine the cell viability using MTT assay (right panel). The growth inhibition rate was calculated based on the control. *P<0.05

Figure 4

The effect of CYD-6-17 on PDPK1 expression and its role in modulating β-catenin expression. (a) The expression of PDPK1 mRNA and protein in RCC cells treated with CYD-6-17. (b) PDPK1 gene promoter activity determined by luciferase reporter assay in RCC cells treated with CYD-6-17. (c) Cells were transfected with different dose of PDPK1 cDNA for 24 h and then treated with CYD-6-17 (0.5 μM) for 48 h. MTT assays were performed to determine cell viability. (d) The mechanism of PDPK1 in modulating GSK3β activity. HK-2 KD and Sor001 cells were transiently transfected with 2.5 μg of vector control, WT, or KI PDPK1 for 24 h, western blot analyses were performed to determine the expression of PDPK1, total GSK3β or phosphorylated GSK3β, and β-catenin levels (left panel). The relative cell number treated with CYD-6-17 (0.5 μM) for 48 h was determined by MTT assay (right panel). *P<0.05

Figure 5

Association of PDPK1 and RCC patient survival who underwent targeted therapies in TCGA data set. PDPK1 mRNA expression data (RNA-Seq) of RCC patient underwent targeted therapies in TCGA was retrieved, and the X-tile was used to generate an optimal cutoff point to dichotomize PDPK1 mRNA level as ‘High' and ‘Low' using a Monte-Carlo P-value <0.05. The Kaplan–Meier method (log-rank test) was used to analyze patient survival after treatments. Sample size (N), median survival (MS), and hazard ratio (HR) are depicted

Figure 6

The efficacy of CYD-6-17 in RCC tumor in vivo. (a) The experimental therapy of CYD-6-17 using ALZET osmotic pumps to deliver 10 mg/kg of drug for 7 days in 786-0 KD subcutaneous xenograft model. Both tumor volume measured by caliper and tumor weight of control or treatment group were depicted. *P<0.05. (b) Immunohistochemistry staining of PDPK1, Ki-67 (proliferation marker), cleaved caspase-3 (apoptosis marker) and p27 (cell cycle arrest marker) in RCC tumors. Quantitative data were shown in the right panel. (c) The total body weight of animal from control and treatment group. (d) A schematic presentation of the mechanism of CYD-6-17 in targeting PDPK1 and its downstream pathways in drug-resistant RCC