Downregulation of Sp1 by Minnelide leads to decrease in HSP70 and decrease in tumor burden of gastric cancer

Abstract

Background: Gastric cancer is the third leading cause of cancer related mortality worldwide with poor survival rates. Even though a number of chemotherapeutic compounds have been used against this disease, stomach cancer has not been particularly sensitive to these drugs. In this study we have evaluated the effect of triptolide, a naturally derived diterpene triepoxide and its water soluble pro-drug Minnelide on several gastric adenocarcinoma cell lines both as monotherapy and in combination with CPT-11.

Methods: Gastric cancer cell lines MKN28 and MKN45 were treated with varying doses of triptolide in vitro. Cell viability was measured using MTT based assay kit. Apoptotic cell death was assayed by measuring caspase activity. Effect of the triptolide pro-drug, Minnelide, was evaluated by implanting the gastric cancer cells subcutaneously in athymic nude mice.

Results: Gastric cancer cell lines MKN28 and MKN45 cells exhibited decreased cell viability and increased apoptosis when treated with varying doses of triptolide in vitro. When implanted in athymic nude mice, treatment with Minnelide reduced tumor burden in both MKN28 derived tumors as well as MKN45 derived tumors. Additionally, we also evaluated Minnelide as a single agent and in combination with CPT-11 in the NCI-N87 human gastric tumor xenograft model.

Conclusion: Our results indicated that the combination of Minnelide with CPT-11 resulted in significantly smaller tumors compared to control. These studies are extremely encouraging as Minnelide is currently undergoing phase 1 clinical trials for gastrointestinal cancers.

Fig 1

A. Triptolide induces apoptotic cell death in gastric cancer cell line. Treatment of moderately differentiated gastric adenocarcinoma cell line, MKN28 with triptolide decreases viability in a dose (25–100nM) as well as time (24–72 hours) dependent manner. Untreated cells served as control. Viability was assessed using CCK-8. B. Treatment of poorly differentiated gastric adenocarcinoma cell line, MKN45 with triptolide decreases viability in a dose (25–100nM) as well as time (24–72 hours) dependent manner. Untreated cells served as control C. Triptolide causes cell death in MKN28 cells via induction of apoptosis, as evidenced by a dose (25–100nM) as well as time (24–48 hours) dependent increase in caspase 3/7 activation. Untreated cells served as control. D. Triptolide causes cell death in MKN45 cells via induction of apoptosis, as evidenced by a dose (25–100nM) as well as time (24–48 hours) dependent increase in caspase 3/7 activation. Untreated cells served as control. E. Western blot showing cleaved PARP as a measure of apoptosis in MKN28 and MKN45 cells after treatment with indicated doses of triptolide. * indicates p value < 0.05 when compared to untreated.

Fig 2

A. Triptolide prodrug Minnelide induces tumor regression in mouse models for gastric cancer. Treatment of mice bearing subcutaneous xenograft tumors derived from MKN45 gastric adenocarcinoma cells with Minnelide (0.21 mg/kg) as well as Minnelide (0.42mg/kg) led to a significant reduction in tumor burden compared to control (saline treated) mice. Tumor volumes were assessed twice weekly. Mice were followed for 21 days. B. Ex-vivo volumes of the MKN45 derived subcutaneous tumors. C. Ex vivo pictures of MKN45 cell derived tumors from mice treated with Minnelide were significantly smaller than the controls. D. Treatment of mice bearing subcutaneous xenograft tumors derived from MKN28 gastric adenocarcinoma cells with Minnelide (0.21 mg/kg) as well as Minnelide (0.42mg/kg) led to a reduction in tumor burden compared to control (saline treated) mice. Tumor volumes were assessed twice weekly. Mice were followed for 42 days. E Ex-vivo volumes of the MKN28 derived subcutaneous tumors. F. Ex vivo pictures of MKN28 cell derived tumors from mice treated with Minnelide were smaller than the controls. G. TUNEL staining of MKN45 as well as MKN28 tumors from mice treated with Minnelide showed significantly higher apoptotic cells than the saline treated mice as evidenced by greater numbers of TUNEL positive cells in the Minnelide treated groups. * indicates p value < 0.05 when compared to untreated.

Fig 3

A. Minnelide and CPT-11 combination is most effective against gastric cancer. Treatment of mice bearing subcutaneous xenograft tumors derived from NCI-N87 gastric adenocarcinoma cell line with a combination of 0.20mg/kg Minnelide and 100mg/kg CPT-11 or 0.30mg/kg Minnelide and 100mg/kg CPT-11 was more effective in reducing tumor burden as compared to vehicle control or Minnelide 0.40mg/kg monotherapy. B. Ex-vivo tumor volumes of the NCI-N87 cell line derived tumors. Combination of lower dose of Minnelide and CPT-11 was more effective in reducing tumor burden compared to vehicle control or Minnelide 0.40 mg/kg alone. * indicates p value < 0.05 when compared to untreated.

Fig 4

A Triptolide induced cell death in gastric cancer is mediated via Sp1. Treatment with 100nM triptolide decreased the mRNA expression of Sp1, HSF1 and HSP70 in MKN28 and MKN45 gastric adenocarcinoma cells in a time (24–48 hours) dependent manner. B. Protein expression of Sp1, HSP70 and HSF1 decreased after 100nM triptolide treatment in both MKN28 and MKN45 cells. C. Treatment of MKN28 and MKN45 cells with mithramycin (a chemical inhibitor of Sp1) led to a reduction in cell viability in a dose (25–200nM) as well as time (24–72 hours) dependent manner similar to triptolide. D. Treatment of MKN28 and MKN45 cells with 100nM mithramycin led to a time (24–48 hours) dependent decrease in the mRNA expression of HSF1 and HSP70, indicating that they lie downstream of Sp1. E. Treatment of MKN45 cells with 100nM mithramycin led to a time (24–48 hours) dependent decrease in the protein expression of HSF1 and HSP70, indicating that they lie downstream of Sp1 in gastric adenocarcinoma cells. F. Overexpression of Sp1 in gastric cancer cell line MKN28 and MKN45 resulted in a rescue from the triptolide induced cell death. * indicates p value < 0.05 when compared to untreated.