Inhibition of epithelial ovarian cancer by Minnelide, a water-soluble pro-drug

doi:10.1016/j.ygyno.2014.08.031

Gynecologic Oncology

Volume 135, Issue 2, November 2014, Pages 318-324

https://doi.org/10.1016/j.ygyno.2014.08.031 Get rights and content

Highlights

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Minnelide is an effective agent against epithelial ovarian cancer in preclinical models.
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Given the promising in vitro and in vivo results, Minnelide should progress to further studies in epithelial ovarian cancer.

Abstract

Objective

Minnelide is a water-soluble pro-drug of triptolide, a natural product. The goal of this study was to evaluate the effectiveness of Minnelide on ovarian cancer growth in vitro and in vivo.

Methods

The effect of Minnelide on ovarian cancer cell proliferation was determined by real time electrical impedance measurements. Multiple mouse models with C200 and A2780 epithelial ovarian cancer cell lines were used to assess the efficacy of Minnelide in inhibiting ovarian cancer growth.

Results

Minnelide decreased cell viability of both platinum sensitive and resistant epithelial ovarian cancer cells in vitro. Minnelide with carboplatin showed additive effects in vitro. Minnelide monotherapy increased the survival of mice bearing established ovarian tumors. Minnelide, in combination with carboplatin and paclitaxel, improved overall survival of mice.

Conclusions

Minnelide is a promising pro-drug for the treatment of ovarian cancer, especially when combined with standard chemotherapy.

Introduction

Ovarian cancer has the highest mortality rate of all of the malignancies of the female genital tract. In the United States 21,980 patients were diagnosed with ovarian cancer in 2014 and 14,270 patients died of their disease [1]. The 5 year survival rate for ovarian cancer is 25% for advanced stage disease, which is when 85% of patients are diagnosed. The poor survival is due to a combination of factors including a lack of effective screening and prevention techniques, late stage at diagnosis and lack of treatment options once a patient has failed initial therapies. The current standard first line treatment for ovarian cancer is a combination of a taxane and platinum agent. While 80% of patients will respond to first line treatment, 20% of patients will be platinum resistant and not respond to standard therapy. There are a number of second line agents approved for the treatment of recurrent ovarian cancer including liposomal doxorubicin, topotecan and gemcitabine; however progression free survival times are generally < 12 months [2]. Given these relatively low response rates there is a need for new drug discovery and more effective ways to treat ovarian cancer.

Triptolide, a diterpenoid triepoxide isolated from the plant Tripterygium wilfordii Hook F, has shown efficacy against several different cancer types including pancreatic, melanoma, neuroblastoma and gastric [3], [4], [5]. Because triptolide is not water soluble, the physical properties of this natural product have limited its clinical development. To overcome solubility problems, triptolide was chemically modified to prepare a prodrug which was named Minnelide [6], [7]. Minnelide or 14-O-phosphonooxymethyltriptolide disodium salt is a white powder which is converted into the parent compound triptolide in the presence of alkaline phosphatase which is common in all tissues in the body [8]. The conversion of triptolide to Minnelide has been shown previously both in vitro and in vivo [6]. This compound has been found to be effective against pancreatic cancer as well as osteosarcoma and non-small cell lung cancer both in vitro and in vivo [6], [9], [10]. Since both pancreatic and ovarian cancers metastasize in the peritoneum, we sought to determine the efficacy of Minnelide in inhibiting ovarian cancer growth.

Section snippets

Reagents

Minnelide was synthesized in the Department of Medicinal Chemistry, University of Minnesota as described by Chugh et al. [6]. Carboplatin was purchased from Calbiochem and Paclitaxel from Sigma Aldrich.

Cell lines and cell culture

Platinum sensitive (A2780) and platinum resistant (C200) ovarian cancer cell lines were obtained from Thomas Hamilton, Fox Chase Cancer Center, Philadelphia. Both cell lines were cultured at 37 °C in the presence of 5% CO₂ in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10%

Minnelide decreases cell viability of ovarian cancer cells in vitro

In the initial experiment, cell viability was determined following treatment with Minnelide. Both A2780 and C200 cells were inhibited in a concentration dependent manner between 10 and 250 nM (Fig. 1). To assess the efficacy of Minnelide on proliferation of ovarian cancer cell lines, we treated A2780 with 50 nM, 100 nM, 200 nM and 500 nM of Minnelide and C200 cells with 200 nM, 500 nM and 1000 nM of Minnelide. Preliminary studies had shown that C200 cells display a multi-drug resistance phenotype and

Discussion

Previous studies have shown that Minnelide, a water soluble analog of triptolide, is effective in pancreatic, gastric and osteosarcoma [9], [10], [3], [5], [13]. We have found that Minnelide is an active agent against ovarian cancer both in vitro and in vivo.

Chugh et al. have shown Minnelide to be highly active and even curative for early pancreatic tumors [6]. Others have shown that Minnelide exhibits anti-proliferative effects in non-small cell lung cancer by promoting apoptosis in vitro and

Conflict of interest statement

Dr. Saluja is the only author with a conflict of interest in that he has Minneamrita, stock options and acts as a consultant for Minnelide.

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Mechanisms of cancer cell death induction by triptolide: A comprehensive overview
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The need for naturally occurring constituents is driven by the rise in the cancer prevalence and the unpleasant side effects associated with chemotherapeutics. Triptolide, the primary active component of “Tripterygium Wilfordii”, has exploited for biological mechanisms and therapeutic potential against various tumors. Based on the recent pre-clinical investigations, triptolide is linked to the induction of death of cancerous cells by triggering cellular apoptosis via inhibiting heat shock protein expression (HSP70), and cyclin dependent kinase (CDKs) by up regulating expression of P21. MKP1, histone methyl transferases and RNA polymerases have all recently identified as potential targets of triptolide in cells. Autophagy, AKT signaling pathway and various pathways involving targeted proteins such as A-disintegrin & metalloprotease-10 (ADAM10), Polycystin-2 (PC-2), dCTP pyro-phosphatase 1 (DCTP1), peroxiredoxin-I (Prx-I), TAK1 binding protein (TAB1), kinase subunit (DNA–PKcs) and the xeroderma-pigmentosum B (XPB or ERCC3) have been exploited. Besides that, triptolide is responsible for enhancing the effectiveness of various chemotherapeutics. In addition, several triptolide moieties, including minnelide and LLDT8, have progressed in investigations on humans for the treatment of cancer. Targeted strategies, such as triptolide conjugation with ligands or triptolide loaded nano-carriers, are efficient techniques to confront toxicities associated with triptolide. We expect and anticipate that advances in near future, regarding combination therapies of triptolide, might be beneficial against cancerous cells.
Triptolide: Reflections on two decades of research and prospects for the future
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Triptolide is a bioactive diterpene triepoxide isolated from Tripterygium wilfordii Hook F, a traditional Chinese medicinal plant whose extracts have been used as anti-inflammatory and immunosuppressive remedies for centuries. Although triptolide and its analogs exhibit potent bioactivities against various cancers, and inflammatory and autoimmune diseases, none of them has been approved to be used in the clinic. This review highlights advances in material sourcing, molecular mechanisms, clinical progress and new drug design strategies for triptolide over the past two decades, along with some prospects for the future course of development of triptolide.
Analysis of the role of geranylgeranyl diphosphate synthase 8 from Tripterygium wilfordii in diterpenoids biosynthesis
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Citation Excerpt :
The genus Tripterygium is known to produce many types of terpenoids, including diterpenoids, sesquiterpenoids, and triterpenoids, of which triptolide, a labdane-type diterpenoid, shows impressive and effective anti-inflammatory, immunosuppressive and antitumor activities, and potential medicinal value for central nervous system diseases (e.g., Parkinson’s and Alzheimer’s diseases) [2–7]. Moreover, several derivatives of triptolide have undergone clinical trials [8–10]. Due to the rich pharmaceutical potential but low content of triptolide in planta, many studies have been targeted toward the elucidation of the triptolide biosynthetic pathway in order to achieve sustainable and reliable triptolide production via synthetic biology strategies [11–13].
Tripterygium wilfordii is known to contain various types of bioactive diterpenoids that exhibit many remarkable activities. Many studies have recently been targeted toward the elucidation of the diterpenoids biosynthetic pathways in attempts to obtain these compounds with a view to solving the dilemma of low yield in plants. However, the short-chain prenyltransferases (SC-PTSs) responsible for the formation of geranylgeranyl diphosphate (GGPP), a crucial precursor for synthesizing the skeleton structures of diterpenoids, have not been characterized in depth. Here, T. wilfordii transcriptome data were used to identify eight putative GGPPSs, including two small subunits of geranyl diphosphate synthase (GPPS.SSU). Of them, GGPPS1, GGPPS7, GGPPS8, GPPS.SSU II and GPPS.SSU were translocated mainly into chloroplasts, and GGPPS8 exhibited the optimal catalytic efficiency with respect to catalyzing the formation of GGPP. In addition, the expression pattern of GGPPS8 was similar to that of downstream terpene synthase genes that are directly correlated with triptolide production in roots, indicating that GGPPS8 was most likely to participate in triptolide biosynthesis in roots among the studied enzymes. GPPS.SSU was inactive alone but interacted with GGPPS1, GGPPS7 and GGPPS8 to change the product from GGPP to GPP. These findings implicate that these candidate genes can be regulated to shift the metabolic flux toward diterpenoid formation, increasing the yields of bioactive diterpenoids in plants.
Triptolide and Its Derivatives as Cancer Therapies
2019, Trends in Pharmacological Sciences
Citation Excerpt :
TPL has attracted considerable attention among researchers due to its efficacy against various diseases, although its clinical potential remains to be fully explored. TPL inhibits cancer cell growth and exhibits preclinical antitumor activity in a variety of cancers including acute myeloid leukemia (AML) [6], breast cancer [7], osteosarcoma [8], ovarian cancer [9], lung cancer [10], prostate cancer [11], neuroblastoma [12], and multiple gastrointestinal cancers (e.g., colon, liver, stomach, and pancreas cancers) [13–16]. In this review, we focus on the mechanism of action and current state of preclinical and clinical development of TPL and its analogs as anticancer agents.
Triptolide, a compound isolated from a Chinese medicinal herb, possesses potent antitumor, immunosuppressive, and anti-inflammatory properties, but is clinically limited due to its poor solubility, bioavailability, and toxicity. Recently, Minnelide, a water-soluble prodrug of triptolide, was shown to have potent antitumor activity in various preclinical cancer models. Minnelide is currently in Phase II clinical trials for treatment of advanced pancreatic cancer, which has fueled increased interest in this promising agent. Here, we review the recent advances in the biological activity of triptolide and its analogs, their mechanisms of actions, and their clinical developments. A special emphasis is given to proteins and pathways within the tumor and stromal compartments that are targeted by triptolide and its analogs as well as the ongoing clinical trials.
Biochemical and computational evaluation of Triptolide-induced cytotoxicity against NSCLC
2018, Biomedicine and Pharmacotherapy
Citation Excerpt :
Chemotherapeutic agents derived from Traditional Chinese Medicine represent a valuable tool for cancer treatment. Triptolide, a natural compound isolated from the Chinese plant Tripterygium wilfordii, has been reported as being effective against various types of cancers including leukemia [26], pancreatic cancer [27,28], breast cancer [29–31], cholangiocarcinoma [11,32],and ovarian cancer [33]. Although it has been studied extensively, the molecular mechanism underlying Triptolide’s activity is still poorly understood and appears to involve multiple signaling pathways.
Triptolide is the major bioactive component isolated from the Chinese Medicinal plant Tripterygium wilfordii. Despite the growing interest and the plethora of reports discussing the pharmacological activity of this diterpenoid, no clear consensus regarding its cellular targets and full mechanism of action has been reached.
In the present work, a combined in vitro and in silico approach was used to evaluate the biological activity of Triptolide on Non-small cell lung cancer (NSCLC). In vitro, Triptolide treatment induced apoptosis in NSCLC cell lines and down-regulated the phosphorylation of AKT, mTOR, and p70S6K. Triptolide also impacted cellular glycolysis as well as the antioxidant response through the impairment of glucose utilization, HKII, glutathione, and NRF2 levels. Molecular docking results examined the possible interactions between Triptolide and AKT and predicted an allosteric binding to AKT-1 structure. Molecular dynamics simulations were further used to evaluate the stability of the complex formed by Triptolide’s best conformer and AKT. These findings provide an insightful approach to the anticancer effect of Triptolide against NSCLC and highlight a possible new role for AKT/mTOR HKII inhibition.
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A valid preclinical tumor model should recapitulate the tumor microenvironment. Immune and stromal components are absent in immunodeficient models of pancreatic cancer. While these components are present in genetically engineered models such as Kras^G12D; Trp53^R172H; Pdx-1Cre (KPC), immense variability in development of invasive disease makes them unsuitable for evaluation of novel therapies. We have generated a novel mouse model of pancreatic cancer by implanting tumor fragments from KPC mice into the pancreas of wild type mice. Three-millimeter tumor pieces from KPC mice were implanted into the pancreas of C57BL/6J mice. Four to eight weeks later, tumors were harvested, and stromal and immune components were evaluated. The efficacy of Minnelide, a novel compound which has been shown to be effective against pancreatic cancer in a number of preclinical murine models, was evaluated. In our model, consistent tumor growth and metastases were observed. Tumors demonstrated intense desmoplasia and leukocytic infiltration which was comparable to that in the genetically engineered KPC model and significantly more than that observed in KPC tumor-derived cell line implantation model. Minnelide treatment resulted in a significant decrease in the tumor weight and volume. This novel model demonstrates a consistent growth rate and tumor-associated mortality and recapitulates the tumor microenvironment. This convenient model is a valuable tool to evaluate novel therapies.

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The research reported in this publication was supported by the NIH grant P30 CA77598 utilizing the Biostatistics and Bioinformatics Core shared resource of the Masonic Cancer Center, University of Minnesota and by the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114.

This research was also supported in part by the Minnesota Ovarian Cancer Alliance (MOCA).

View full text

Inhibition of epithelial ovarian cancer by Minnelide, a water-soluble pro-drug☆

Highlights

Abstract

Objective

Methods

Results

Conclusions

Introduction

Section snippets

Reagents

Cell lines and cell culture

Minnelide decreases cell viability of ovarian cancer cells in vitro

Discussion

Conflict of interest statement

Surgery

Cancer Lett

Microvasc Res

J Surg Res

National Comprehensive Cancer Network Treatment Guidelines

Triptolide inhibits the growth and metastasis of solid tumors

Mol Cancer Ther

Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70

Cancer Res

A preclinical evaluation of Minnelide as a therapeutic agent against pancreatic cancer

Sci Transl Med