TP53 and Let-7a micro-RNA Regulate K-Ras Activity in HCT116 Colorectal Cancer Cells

Recent reports have indicated that KRAS and TP53 mutations predict response to therapy in colorectal cancer. However, little is known about the relationship between these two common genetic alterations. Micro-RNAs (miRNAs), a class of noncoding RNA implicated in cellular processes, have been increasingly linked to KRAS and TP53. We hypothesized that lethal-7a (let-7a) miRNA regulates KRAS through TP53. To investigate the relationship between KRAS, TP53, and let-7a, we used HCT116 KRAS^mut human colorectal cancer cells with four different genotypic modifications in TP53 (TP53^−/−, TP53^+/−, TP53^mut/+, and TP53^mut/−). Using these cells we observed that K-Ras activity was higher in cells with mutant or knocked out TP53 alleles, suggesting that wild-type TP53 may suppress K-Ras activity. Let-7a was present in HCT116 KRAS^mut cells, though there was no correlation between let-7a level and TP53 genotype status. To explore how let-7a may regulate K-Ras in the different TP53 genotype cells we used let-7a inhibitor and demonstrated increased K-Ras activity across all TP53, thus corroborating prior reports that let-7a regulates K-Ras. To assess potential clinical implications of this regulatory network, we examined the influence of TP53 genotype and let-7a inhibition on colon cancer cell survival following chemoradiation therapy (CRT). We observed that cells with complete loss of wild-type TP53 alleles (^−/− or ^−/mut) were resistant to CRT following treatment with 5-fluorouracil and radiation. Further increase in K-Ras activity with let-7a inhibition did not impact survival in these cells. In contrast, cells with single or double wild-type TP53 alleles were moderately responsive to CRT and exhibited resistance when let-7a was inhibited. In summary, our results show a complex regulatory system involving TP53, KRAS, and let-7a. Our results may provide clues to understand and target these interactions in colorectal cancer.     

A Novel Synthetic Microtubule Inhibitor,MPT0B214 Exhibits Antitumor Activity in Human Tumor Cells through Mitochondria-Dependent Intrinsic Pathway

Agents that interfere with mitotic progression by disturbing microtubule dynamics are commonly used for cancer treatment. Previously, a series of aroylquinolone regioisomers as novel microtubule inhibitors were discovered. One of these new compounds, MPT0B214 inhibited tubulin polymerization through strongly binding to the tubulin’s colchicine-binding site and had cytotoxic activity in a variety of human tumor cell lines. After treatment with MPT0B214, KB cells were arrested in the G₂-M phase before cell death occurred, which were associated with upregulation of cyclin B1, dephosphorylation of Cdc2, phosphorylation of Cdc25C and elevated expression of the mitotic marker MPM-2. Furthermore, the compound induced apoptotic cell death through mitochondria/caspase 9-dependent pathway. Notably, several KB-derived multidrug-resistant cancer cell lines were also sensitive to MPT0B214 treatment. These findings showed that MPT0B214 is a potential compound in the treatment of various malignancies.     

In Vitro and In Vivo Metabolism and Inhibitory Activities of Vasicine,a Potent Acetylcholinesterase and Butyrylcholinesterase Inhibitor

Vasicine (VAS), a potential natural cholinesterase inhibitor, exhibited promising anticholinesterase activity in preclinical models and has been in development for treatment of Alzheimer’s disease. This study systematically investigated the in vitro and in vivo metabolism of VAS in rat using ultra performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry. A total of 72 metabolites were found based on a detailed analysis of their ¹H- NMR and ¹³C NMR data. Six key metabolites were isolated from rat urine and elucidated as vasicinone, vasicinol, vasicinolone, 1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-yl hydrogen sulfate, 9-oxo-1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-yl hydrogen sulfate, and 1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-β-D-glucuronide. The metabolic pathway of VAS in vivo and in vitro mainly involved monohydroxylation, dihydroxylation, trihydroxylation, oxidation, desaturation, sulfation, and glucuronidation. The main metabolic soft spots in the chemical structure of VAS were the 3-hydroxyl group and the C-9 site. All 72 metabolites were found in the urine sample, and 15, 25, 45, 18, and 11 metabolites were identified from rat feces, plasma, bile, rat liver microsomes, and rat primary hepatocyte incubations, respectively. Results indicated that renal clearance was the major excretion pathway of VAS. The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of VAS and its main metabolites were also evaluated. The results indicated that although most metabolites maintained potential inhibitory activity against AChE and BChE, but weaker than that of VAS. VAS undergoes metabolic inactivation process in vivo in respect to cholinesterase inhibitory activity.     

KCN1, a Novel Synthetic Sulfonamide Anticancer Agent: In Vitro and In Vivo Anti-Pancreatic Cancer Activities and Preclinical Pharmacology

The purpose of the present study was to determine the in vitro and in vivo anti-cancer activity and pharmacological properties of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1. In the present study, we investigated the in vitro activity of KCN1 on cell proliferation and cell cycle distribution of pancreatic cancer cells, using the MTT and BrdUrd assays, and flow cytometry. The in vivo anti-cancer effects of KCN1 were evaluated in two distinct xenograft models of pancreatic cancer. We also developed an HPLC method for the quantitation of the compound, and examined its stability in mouse plasma, plasma protein binding, and degradation by mouse S9 microsomal enzymes. Furthermore, we examined the pharmacokinetics of KCN1 following intravenous or intraperitoneal injection in mice. Results showed that, in a dose-dependent manner, KCN1 inhibited cell growth and induced cell cycle arrest in human pancreatic cancer cells in vitro, and showed in vivo anticancer efficacy in mice bearing Panc-1 or Mia Paca-2 tumor xenografts. The HPLC method provided linear detection of KCN1 in all of the matrices in the range from 0.1 to 100 µM, and had a lower limit of detection of 0.085 µM in mouse plasma. KCN1 was very stable in mouse plasma, extensively plasma bound, and metabolized by S9 microsomal enzymes. The pharmacokinetic studies indicated that KCN1 could be detected in all of the tissues examined, most for at least 24 h. In conclusion, our preclinical data indicate that KCN1 is a potential therapeutic agent for pancreatic cancer, providing a basis for its future development.     

The Novel Deacetylase Inhibitor AR-42 Demonstrates Pre-Clinical Activity in B-Cell Malignancies In Vitro and In Vivo

Background

While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies.

Principal Findings

In mantle cell lymphoma (JeKo-1), Burkitt''s lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC₅₀ (50% growth inhibitory concentration) of AR-42 is 0.61 µM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC₅₀ (concentration lethal to 50%) of AR-42 is 0.76 µM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity.

Conclusions/Significance

Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.     

Suberoylanilide Hydroxamic Acid,an Inhibitor of Histone Deacetylase,Enhances Radiosensitivity and Suppresses Lung Metastasis in Breast Cancer In Vitro and In Vivo

Triple-negative breast cancer (TNBC), defined by the absence of an estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, is associated with an early recurrence of disease and poor outcome. Furthermore, the majority of deaths in breast cancer patients are from metastases instead of from primary tumors. In this study, MCF-7 (an estrogen receptor-positive human breast cancer cell line), MDA-MB-231 (a human TNBC cell line) and 4T1 (a mouse TNBC cell line) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with suberoylanilide hydroxamic acid (SAHA, an inhibitor of histone deacetylase (HDAC)) and to determine the underlying mechanisms of these effects in vitro and in vivo. We also evaluated the ability of SAHA to inhibit the metastasis of 4T1 cells. We found that IR combined with SAHA showed increased therapeutic efficacy when compared with either treatment alone in MCF-7, MDA-MB-231 and 4T1 cells. Moreover, the combined treatment enhanced DNA damage through the inhibition of DNA repair proteins. The combined treatment was induced primarily through autophagy and ER stress. In an orthotopic breast cancer mouse model, the combination treatment showed a greater inhibition of tumor growth. In addition, SAHA inhibited the migration and invasion abilities of 4T1 cells and inhibited breast cancer cell migration by inhibiting the activity of MMP-9. In an in vivo experimental metastasis mouse model, SAHA significantly inhibited lung metastasis. SAHA not only enhances radiosensitivity but also suppresses lung metastasis in breast cancer. These novel findings suggest that SAHA alone or combined with IR could serve as a potential therapeutic strategy for breast cancer.     

Anti-Hepatoma Activity of a Novel Compound Glaucocalyxin H In Vivo and In Vitro

Glaucocalyxin H (GLH) is a new compound isolated from a traditional Chinese medical herb Isodon japonica var. glaucocalyx which has been used for folk medicine. This study was carried out for the first time to investigate the potential role of GLH in anti-hepatoma activity and underlying mechanisms in it. GLH could inhibit the growth of tumor in mice and induce HepG₂ cells to death as assessed by the tumor reduction assay, toxic assay, morphological change, and survival rate assay. Many antitumor drugs originated from plants could inhibit the growth of tumor by inducing cells to apoptosis. The morphological changes of HepG₂ cells treated with different concentrations of GLH under fluorescence and electron microscope and apoptotic rates were detected to verify its effect on apoptosis. As shown in the study, GLH could induce HepG₂ cells to apoptosis in a dose-dependent manner. Bcl₂ and Bax proteins played important roles in apoptosis and the disequilibrium between Bcl₂ and Bax might result in apoptosis. The expression of Bax protein was upregulated and Bcl₂ protein was downregulated in HepG₂ cells treated with GLH assessed by Western blotting, and they were in a dose-dependent manner. Taken together, GLH can inhibit the growth of hepatoma cells in vivo and in vitro by inducing cell apoptosis due to the decreased Bcl₂ and increased Bax proteins suggesting that GLH could be a potential candidate as an anti-hepatoma agent for the therapeutic treatment of hepatoma.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0227-3) contains supplementary material, which is available to authorized users.KEY WORDS: apoptosis, Glaucocalyxin H, hepatoma, HepG₂ cell     

Effectiveness of the Histone Deacetylase Inhibitor (S)-2 against LNCaP and PC3 Human Prostate Cancer Cells

Histone deacetylase inhibitors (HDACi) represent a promising class of epigenetic agents with anticancer properties. Here, we report that (S)-2, a novel hydroxamate-based HDACi, shown previously to be effective against acute myeloid leukemia cells, was also a potent inducer of apoptosis/differentiation in human prostate LNCaP and PC3 cancer cells. In LNCaP cells (S)-2 was capable of triggering H3/H4 histone acetylation, H2AX phosphorylation as a marker of DNA damage and producing G₀/G₁ cell cycle arrest. Consistently, (S)-2 led to enhanced expression of both the protein and mRNA p21 levels in LNCaP cells but, contrary to SAHA, not in normal non-tumorigenic prostate PNT1A cells. Mechanistic studies demonstrated that (S)-2-induced apoptosis in LNCaP cells developed through the cleavage of pro-caspase 9 and 3 and of poly(ADP-ribose)-polymerase accompanied by the dose-dependent loss of mitochondrial membrane potential. Indeed, the addition of the pan-caspase inhibitor Z-VAD-fmk greatly reduced drug-mediated apoptosis while the antioxidant N-acetyl-cysteine was virtually ineffective. Importantly, preliminary data with nude mice xenografted with LNCaP cells showed that (S)-2 prompted a decrease in the tumor volume and an increase in H2AX phosphorylation within the cancer cells. Moreover, the highly metastatic prostate cancer PC3 cells were also sensitive to (S)-2 that: i) induced growth arrest and moderate apoptosis; ii) steered cells towards differentiation and neutral lipid accumulation; iii) reduced cell invasiveness potential by decreasing the amount of MMP-9 activity and up-regulating TIMP-1 expression; and iv) inhibited cell motility and migration through the Matrigel. Overall, (S)-2 has proven to be a powerful HDACi capable of inducing growth arrest, cell death and/or differentiation of LNCaP and PC3 prostate cancer cells and, due to its low toxicity and efficacy in vivo, might also be of clinical interest to support conventional prostate cancer therapy.     

GMP-Compliant,Large-Scale Expanded Allogeneic Natural Killer Cells Have Potent Cytolytic Activity against Cancer Cells In Vitro and In Vivo

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3⁺ T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3⁻CD16⁺CD56⁺ NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.     

Combining mTOR Inhibition with Radiation Improves Antitumor Activity in Bladder Cancer Cells In Vitro and In Vivo: A Novel Strategy for Treatment

Purpose

Radiation therapy for invasive bladder cancer allows for organ preservation but toxicity and local control remain problematic. As such, improving efficacy of treatment requires radiosensitization of tumor cells. The aim of study is to investigate if the mammalian Target of Rapamycin (mTOR), a downstream kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT survival pathway, may be a target for radiation sensitization.

Experimental Design

Clonogenic assays were performed using 6 bladder cancer cell lines (UM-UC3, UM-UC5, UM-UC6, KU7, 253J-BV, and 253-JP) in order to examine the effects of ionizing radiation (IR) alone and in combination with RAD001, an mTOR inhibitor. Cell cycle analysis was performed using flow cytometry. In vivo, athymic mice were subcutaneously injected with 2 bladder cancer cell lines. Treatment response with RAD001 (1.5 mg/kg, daily), fractionated IR (total 9Gy = 3Gy×3), and combination of RAD001 and IR was followed over 4 weeks. Tumor weight was measured at experimental endpoint.

Results

Clonogenic assays revealed that in all bladder cell lines tested, an additive effect was observed in the combined treatment when compared to either treatment alone. Our data indicates that this effect is due to arrest in both G1 and G2 phases of cell cycle when treatments are combined. Furthermore, our data show that this arrest is primarily regulated by changes in levels of cyclin D1, p27 and p21 following treatments. In vivo, a significant decrease in tumor weight was observed in the combined treatment compared to either treatment alone or control.

Conclusions

Altering cell cycle by inhibiting the mTOR signaling pathway in combination with radiation have favorable outcomes and is a promising therapeutic modality for bladder cancer.     

MicroRNA Let-7a Inhibits Proliferation of Human Prostate Cancer Cells In Vitro and In Vivo by Targeting E2F2 and CCND2

Background

Previous work has shown reduced expression levels of let-7 in lung tumors. But little is known about the expression or mechanisms of let-7a in prostate cancer. In this study, we used in vitro and in vivo approaches to investigate whether E2F2 and CCND2 are direct targets of let-7a, and if let-7a acts as a tumor suppressor in prostate cancer by down-regulating E2F2 and CCND2.

Methodology/Principal

Findings Real-time RT-PCR demonstrated that decreased levels of let-7a are present in resected prostate cancer samples and prostate cancer cell lines. Cellular proliferation was inhibited in PC3 cells and LNCaP cells after transfection with let-7a. Cell cycle analysis showed that let-7a induced cell cycle arrest at the G1/S phase. A dual-luciferase reporter assay demonstrated that the 3′UTR of E2F2 and CCND2 were directly bound to let-7a and western blotting analysis further indicated that let-7a down-regulated the expression of E2F2 and CCND2. Our xenograft models of prostate cancer confirmed the capability of let-7a to inhibit prostate tumor development in vivo.

Conclusions/Significance

These findings help to unravel the anti-proliferative mechanisms of let-7a in prostate cancer. Let-7a may also be novel therapeutic candidate for prostate cancer given its ability to induce cell-cycle arrest and inhibit cell growth, especially in hormone-refractory prostate cancer.     

In Vitro and In Vivo Antitumor Activity of a Novel Semisynthetic Derivative of Cucurbitacin B

Lung cancer is the most deadly type of cancer in humans, with non-small-cell lung cancer (NSCLC) being the most frequent and aggressive type of lung cancer showing high resistance to radiation and chemotherapy. Despite the outstanding progress made in anti-tumor therapy, discovering effective anti-tumor drugs is still a challenging task. Here we describe a new semisynthetic derivative of cucurbitacin B (DACE) as a potent inhibitor of NSCLC cell proliferation. DACE arrested the cell cycle of lung epithelial cells at the G2/M phase and induced cell apoptosis by interfering with EGFR activation and its downstream signaling, including AKT, ERK, and STAT3. Consistent with our in vitro studies, intraperitoneal application of DACE significantly suppressed the growth of mouse NSCLC that arises from type II alveolar pneumocytes due to constitutive expression of a human oncogenic c-RAF kinase (c-RAF-1-BxB) transgene in these cells. Taken together, these findings suggest that DACE is a promising lead compound for the development of an anti-lung-cancer drug.     

Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo

Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (-/-) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity.     

The Histone Deacetylase Inhibitor Sodium Butyrate Promotes Cell Death and Differentiation and Reduces Neurosphere Formation in Human Medulloblastoma Cells

Increasing evidence suggests that alterations in epigenetic mechanisms regulating chromatin state play a role in the pathogenesis of medulloblastoma (MB), the most common malignant brain tumor of childhood. Histone deacetylase (HDAC) inhibitors, which increase chromatin relaxation, have been shown to display anticancer activities. Here we show that the HDAC inhibitor sodium butyrate (NaB) markedly increases cell death and reduces colony formation in human MB cell lines. In addition, NaB increased the mRNA expression of Gria2, a neuronal differentiation marker, in D283 and DAOY cells and reduced the number of neurospheres in D283 cell cultures. Finally, NaB reduced the viability of D283 cells when combined with etoposide. These data show that NaB displays pronounced inhibitory effects on the survival of human MB cells and suggest that NaB might potentiate the effects of etoposide. In addition, our study suggests that HDAC inhibition might promote the neuronal differentiation of MB cells and provides the first evidence that an HDAC inhibitor might suppress the expansion or survival of MB cancer stem cells.     

A Novel Compound NSC745885 Exerts an Anti-Tumor Effect on Tongue Cancer SAS Cells In Vitro and In Vivo

Objective

Oral squamous cell carcinoma (OSCC) is a prevalent cancer, especially in developing countries. Anthracyclines and their anthraquinone derivatives, such as doxorubicin, exhibit a cell growth inhibitory effect and have been used as anti-cancer drugs for many years. However, the cardiotoxicity of anthracycline antibiotics is a major concern in their clinical application. NSC745885 is a novel compound synthesized from 1,2-diaminoanthraquinone, which subsequently reacts with thionyl chloride and triethylamine. The present study aimed to investigate the anti-oral cancer potential and the safety of NSC745885.

Methods

We investigated the anti-cancer potential of NSC745885 in oral squamous carcinoma cell lines and in an in vivo oral cancer xenograft mouse model. The expression of apoptotic related genes were evaluated by real-time RT-PCR and western bloting, and the in vivo assessment of apoptotic marker were measured by immunohistochemical staining. The anti-tumor efficiency and safety between doxorubicin and NSC745885 were also compared.

Results

Our results demonstrated that NSC745885 exhibits anti-oral cancer activity through the induction of apoptosis in cancer cells and in tumor-bearing mice, and this treatment did not induce marked toxicity in experimental mice. This compound also exhibits a comparable anti-tumor efficiency and a higher safety in experimental mice when compared to doxorubicin.

Conclusions

The data of this study provide evidence for NSC745885 as a potential novel therapeutic drug for the treatment of human OSCC.