Thymoquinone induces apoptosis and DNA damage in 5-Fluorouracil-resistant colorectal cancer stem/progenitor cells.

The high recurrence rates of colorectal cancer have been associated with a small population of cancer stem cells (CSCs) that are resistant to the standard chemotherapeutic drug, 5-fluorouracil (5FU). Thymoquinone (TQ) has shown promising antitumor properties on numerous cancer systems both in vitro and in vivo; however, its effect on colorectal CSCs is poorly established. Here, we investigated TQ's potential to target CSCs in a three-dimensional (3D) sphere-formation assay enriched for a population of colorectal cancer stem/progenitor cells. Our results showed a significant decrease in self-renewal potential of CSC populations enriched from 5FU-sensitive and resistant HCT116 cells at 10-fold lower concentrations when compared to 2D monolayers. TQ decreased the expression levels of colorectal stem cell markers CD44 and Epithelial Cell Adhesion Molecule EpCAM and proliferation marker Ki67 in colonospheres derived from both cell lines and reduced cellular migration and invasion. Further investigation revealed that TQ treatment led to increased TUNEL positivity and a dramatic increase in the amount of the DNA damage marker gamma H2AX particularly in 5FU-resistant colonospheres, suggesting that the diminished sphere forming ability in TQ-treated colonospheres is due to induction of DNA damage and apoptotic cell death. The intraperitoneal injection of TQ in mice inhibited tumor growth of spheres derived from 5FU-sensitive and 5FU-resistant HCT116 cells. Furthermore, TQ induced apoptosis and inhibited NF-κB and MEK signaling in mouse tumors. Altogether, our findings document TQ's effect on colorectal cancer stem-like cells and provide insights into its underlying mechanism of action.

Natural and synthetic retinoids in preclinical colorectal cancer models.

Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality worldwide. Although targeted therapy in combination with chemotherapy in CRC prolongs the overall survival of patients with metastatic disease, acquired resistance and relapse hinder their clinical benefits. Moreover, patients with some specific genetic profile are unlikely to benefit from targeted therapy, suggesting the need for safe and effective treatment strategies. Retinoids, comprising of natural and synthetic analogs, are a class of chemical compounds that regulate cellular proliferation, differentiation, and cell death. Retinoids have been used in the clinic for several leukemias and solid tumors, either as single agents or in combination therapy. Furthermore, retinoids have shown potent chemotherapeutic and chemopreventive properties in different cancer models, including CRC. In this review, we summarize the major preclinical findings in CRC in which natural and synthetic retinoids showed promising antitumor activities and stress on the proposed mechanisms of action. Understanding of the retinoids' antitumor mechanisms would provide insights to support and warrant their development in the management of CRC.

The synthetic retinoid ST1926 attenuates prostate cancer growth and potentially targets prostate cancer stem-like cells.

Retinoids are vitamin A derivatives that regulate crucial biological processes such as cellular proliferation, apoptosis, and differentiation. The use of natural retinoids in cancer therapy is limited due to their toxicity and the acquired resistance by cancer cells. Therefore, synthetic retinoids were developed, such as the atypical adamantyl retinoid ST1926 that provides enhanced bioavailability and reduced toxicity. We have assessed the in vitro and in vivo antitumor properties and mechanism of action of ST1926 in targeting cancer stem-like cells population of human prostate cancer (PCa) cell lines, DU145 and PC3, and mouse PCa cell lines, PLum-AD and PLum-AI. We demonstrated that ST1926 substantially reduced proliferation of PCa cells and induced cell cycle arrest, p53-independent apoptosis, and early DNA damage. It also decreased migration and invasion of PCa cells and significantly reduced prostate spheres formation ability in vitro denoting sufficient eradication of the self-renewal ability of the highly androgen-resistant cancer stem cells. Importantly, ST1926 potently inhibited PCa tumor growth and progression in vivo. Our results highlight the potential of ST1926 in PCa therapy and warrant its clinical development.

Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α.

Despite advances in therapeutic strategies, colorectal cancer (CRC) remains the third cause of cancer-related deaths with a relatively low survival rate. Resistance to standard chemotherapy represents a major hurdle in disease management; therefore, developing new therapeutic agents demands a thorough understanding of their mechanisms of action. One of these compounds is ST1926, an adamantyl retinoid that has shown potent antitumor activities in several human cancer models. Here, we show that ST1926 selectively suppressed the proliferation of CRC cells while sparing normal counterparts, and significantly reduced tumor volume in a xenograft cancer mouse model. Next, we investigated the effects of ST1926 in CRC cells and observed early DNA damage, S-phase arrest, dissipation of mitochondrial membrane potential, and apoptosis induction, in a p53 and p21-independent manner. To address the underlying mechanism of resistance to ST1926, we generated ST1926-resistant HCT116 cells and sequenced DNA polymerase α (POLA1), which was reported to be a direct target to the drug's parent molecule, CD437. We identified similar mutations in POLA1 that conferred resistance to ST1926 and CD437. These mutations were absent in 5-fluorouracil-resistant HCT116 cells, clearly validating the specificity of these mutations to the lack of DNA damage and acquired resistance to ST1926. ST1926 also inhibited POLA1 activity and reduced its protein expression levels. Further, in silico analysis of normal and malignant tissue expression data demonstrated that POLA1 levels are elevated in CRC cells and tissues compared to normal counterparts as well as to other cancer types. Our findings highlight previously uncharacterized mechanisms of action of ST1926 in CRC and suggest that elevated POLA1 expression is a pertinent molecular feature and an attractive target in CRC.

Antitumor activity of the synthetic retinoid ST1926 on primary effusion lymphoma in vitro and in vivo models.

Primary effusion lymphoma (PEL) is a rare B-cell neoplasm, associated with Kaposi sarcoma-associated herpes virus/human herpes virus-8 (KSHV/HHV-8), arising as malignant effusions in body cavities. PEL cells do not harbor conventional genetic cancer mutations; however, their oncogenesis is mainly attributed to HHV-8 latent genes. Treatment strategies are inefficient resulting in poor prognosis of PEL patients, stressing the need for new effective therapy. ST1926 is a synthetic retinoid with favorable antitumor properties and no cross-resistance with the natural retinoid, all-trans retinoic acid. ST1926 has shown potent apoptotic activities on a variety of solid tumors and hematologic malignancies in in vitro and in vivo models. In the present study we elucidated the antitumor activities and underlying molecular mechanism of ST1926 using in vitro, ex vivo, and in vivo PEL preclinical models. ST1926, at sub­micromolar concentrations, displayed potent antiproliferative effects on PEL cell lines and malignant ascites. Furthermore, ST1926 treatment of PEL cells and ascites resulted in their accumulation in the sub-G1 region, S phase cell cycle arrest, early DNA damage, PARP cleavage and p53 activation including the upregulation of its target genes p21 and Bax. However, ST1926 did not significantly modulate HHV-8 latent viral transcripts. Importantly, ST1926 delayed formation of ascites and enhanced survival of PEL mice. These results highlight the therapeutic potential of ST1926 in combination with drugs that target HHV-8 in PEL patients.

Antitumor Effect of the Atypical Retinoid ST1926 in Acute Myeloid Leukemia and Nanoparticle Formulation Prolongs Lifespan and Reduces Tumor Burden of Xenograft Mice.

Acute myeloid leukemia (AML) is one of the most frequent types of blood malignancies. It is a complex disorder of undifferentiated hematopoietic progenitor cells. The majority of patients generally respond to intensive therapy. Nevertheless, relapse is the major cause of death in AML, warranting the need for novel treatment strategies. Retinoids have demonstrated potent differentiation and growth regulatory effects in normal, transformed, and hematopoietic progenitor cells. All-trans retinoic acid (ATRA) is the paradigm of treatment in acute promyelocytic leukemia, an AML subtype. The majority of AML subtypes are, however, resistant to ATRA. Multiple synthetic retinoids such as ST1926 recently emerged as potent anticancer agents to overcome such resistance. Despite its lack of toxicity, ST1926 clinical development was restricted due to its limited bioavailability and rapid excretion. Here, we investigate the preclinical efficacy of ST1926 and polymer-stabilized ST1926 nanoparticles (ST1926-NP) in AML models. We show that sub-µmol/L concentrations of ST1926 potently and selectively inhibited the growth of ATRA-resistant AML cell lines and primary blasts. ST1926 induced-growth arrest was due to early DNA damage and massive apoptosis in AML cells. To enhance the drug's bioavailability, ST1926-NP were developed using Flash NanoPrecipitation, and displayed comparable anti-growth activities to the naked drug in AML cells. In a murine AML xenograft model, ST1926 and ST1926-NP significantly prolonged survival and reduced tumor burden. Strikingly, in vivo ST1926-NP antitumor effects were achieved at four fold lower concentrations than the naked drug. These results highlight the promising use of ST1926 in AML therapy and encourage its further development. Mol Cancer Ther; 16(10); 2047-57. ©2017 AACR.

Antitumor activities of the synthetic retinoid ST1926 in two-dimensional and three-dimensional human breast cancer models.

Despite recent advances in chemotherapy, aggressive and metastatic breast cancers remain refractory to targeted therapy and the development of novel drugs is urgently needed. Retinoids are crucial regulators of cellular proliferation, differentiation, and cell death, and have shown potent chemotherapeutic and chemopreventive properties. The major drawback of the use of all-trans retinoic acid (ATRA) in cancer therapy is disease relapse. Therefore, synthetic retinoids, specifically ST1926, have emerged as potent anticancer agents. Given the importance of the microenvironment in modulating the response of cancer cells to chemotherapeutic drugs, we investigated the antitumor activities of ST1926 in two-dimensional (2D) and different three-dimensional (3D) human breast cancer models and compared them with ATRA. We have shown that in 2D cell culture models, ATRA-resistant MCF-7 and MDA-MB-231 cells were sensitive to ST1926 at submicromolar concentrations that spared the 'normal-like' breast epithelial cells. ST1926 induced apoptosis and S-phase arrest, caused DNA damage, and downregulated the Wnt/ß-catenin pathway in breast cancer cells in 2D and 3D cell culture models. ST1926-mediated growth inhibition was independent of the retinoid receptor-signaling pathway. Long-term treatments with low submicromolar ST1926 concentrations reduced the anchorage-independent growth and decreased the sphere-forming ability of breast cancer progenitor cells in the sphere formation assay. Furthermore, ST1926 potently induced cell death of breast cancer cells under 3D conditions and spared the lumen-forming ability of normal-like breast epithelial cells. In tested 3D models, ATRA had minimal effects on the growth of breast cancer cells compared with ST1926. In summary, our results highlight the therapeutic potential of ST1926 in breast cancer and warrant its further clinical development.

Preclinical efficacy of the synthetic retinoid ST1926 for treating adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). The HTLV-1 oncoprotein Tax plays an important role in ATL pathogenesis. ATL carries a poor prognosis due to chemotherapy resistance, stressing the need for alternative therapies. Here, we investigate the preclinical efficacy of the synthetic retinoid ST1926 in ATL and peripheral T-cell lymphomas. Clinically achievable concentrations of ST1926 induced a dramatic inhibition of cell proliferation in malignant T-cell lines and primary ATL cells with minimal effect on resting or activated normal lymphocytes. ST1926 induced apoptosis, DNA damage, and upregulation of p53 proteins in malignant T cells, whereas it caused an early downregulation of Tax proteins in HTLV-1-positive cells. In murine ATL, oral treatment with ST1926 prolonged survival and reduced leukemia cell infiltration, white blood cell counts, and spleen mass. In spleens of ST1926-treated animals, p53 and p21 proteins were upregulated, poly (ADP-ribose) polymerase was cleaved, and Tax transcripts were reduced. These results highlight the promising use of ST1926 as a targeted therapy for ATL.

A new mechanism of SOX9 action to regulate PKCalpha expression in the intestine epithelium.

Variations of protein kinase C (PKC) expression greatly influence the proliferation-to-differentiation transition (PDT) of intestinal epithelial cells and might have an important impact on intestinal tumorigenesis. We demonstrate here that the expression of PKCalpha in proliferating intestinal epithelial cells is repressed both in vitro and in vivo by the SOX9 transcription factor. This repression does not require DNA binding of the SOX9 high-mobility group (HMG) domain but is mediated through a new mechanism of SOX9 action requiring the central and highly conserved region of SOXE members. Because SOX9 expression is itself upregulated by Wnt-APC signaling in intestinal epithelial cells, the present study points out this transcription factor as a molecular link between the Wnt-APC pathway and PKCalpha. These results provide a potential explanation for the decrease of PKCalpha expression in colorectal cancers with constitutive activation of the Wnt-APC pathway.