Development of Novel ß-Carboline/Furylmalononitrile Hybrids as Type I/II Photosensitizers with Chemo-Photodynamic Therapy and Minimal Toxicity.

Chemo-photodynamic therapy is a treatment method that combines chemotherapy and photodynamic therapy and has demonstrated significant potential in cancer treatment. However, the development of chemo-photodynamic therapeutic agents with fewer side effects still poses a challenge. Herein, we designed and synthesized a novel series of ß-carboline/furylmalononitrile hybrids 10a-i and evaluated their chemo-photodynamic therapeutic effects. Most of the compounds were photodynamically active and exhibited cytotoxic effects in four cancer cells. In particular, 10f possessed type-I/II photodynamic characteristics, and its 1O2 quantum yield increased by 3-fold from pH 7.4 to 4.5. Most interestingly, 10f exhibited robust antiproliferative effects by tumor-selective cytotoxicities and hypoxic-overcoming phototoxicities. In addition, 10f generated intracellular ROS and induced hepatocellular apoptosis, mitochondrial damage, and autophagy. Finally, 10f demonstrated extremely low acute toxicity (LD50 = 1415 mg/kg) and a high tumor-inhibitory rate of 80.5% through chemo-photodynamic dual therapy. Our findings may provide a promising framework for the design of new photosensitizers for chemo-photodynamic therapy.

Upregulation of long non-coding RNA MYU promotes proliferation, migration and invasion of esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumour type of the digestive system. Long non-coding RNA (lncRNA) c-Myc upregulated (MYU), also known as VPS9 domain-containing 1 antisense 1, was recently discovered. However, the expression of lncRNA MYU in ESCC and its role in tumour progression have remained elusive. In the present study, the expression of lncRNA MYU, Ki-67 and the epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin in ESCC tissues was detected by reverse transcription-quantitative PCR. The expression of Ki-67, E-cadherin and Vimentin in ESCC tissues was also detected by immunohistochemistry. A small interfering RNA plasmid was employed to establish a TE-2 cell line with knockdown on lncRNA MYU. The results indicated that the expression of lncRNA MYU was higher in ESCC tissues than in normal adjacent tissues and that upregulation of lncRNA MYU was a potential biomarker for poor prognosis. The results also suggested that the expression levels of lncRNA MYU were correlated with the histological grade, lymph node metastasis and TNM stage (P<0.05). Silencing of lncRNA MYU expression inhibited the proliferation, migration and invasion, while the expression of lncRNA MYU increased as cell proliferation increased. In addition, the mRNA expression of Vimentin and Ki-67 was decreased in TE-2 cells after lncRNA MYU was knocked down, while E-cadherin mRNA expression was elevated. In conclusion, the present results indicated that lncRNA MYU may regulate the proliferation, migration and invasion of ESCC cells, and may serve as a prognostic biomarker for ESCC.

Novel Phenylmethylenecyclohexenone Derivatives as Potent TrxR Inhibitors Display High Antiproliferative Activity and Induce ROS, Apoptosis, and DNA Damage.

The natural product piperlonguminine (PL) has been shown to exert potential anticancer activity against several types of cancer via elevation of reactive oxidative species (ROS). However, the application of PL has been limited due to its poor water solubility and moderate activity. To improve PL's potency, we designed and synthesized a series of 17 novel phenylmethylenecyclohexenone derivatives and evaluated their pharmacological properties. Most of them exerted antiproliferative activities against four cancer cell lines with IC50 values lower than PL. Among these, compound 10 e not only showed good water solubility and exerted the most potent antiproliferative activity against HGC27 cells (IC50 =0.76 µM), which was 10-fold lower than PL (IC50 =7.53 µM), but also exhibited lower cytotoxicity in human normal gastric epithelial cells GES-1 compared with HGC27 cells. Mechanistically, compound 10 e inhibited thioredoxin reductase (TrxR) activity, increased ROS levels, and diminished mitochondrial transmembrane potential (MTP) in HGC27 cells. Furthermore, 10 e also induced G2 /M cell-cycle arrest, and triggered cancer cell apoptosis through the regulation of apoptotic proteins. Finally, 10 e promoted DNA damage in HGC27 cells via the activation of the H2AX(S139ph) and p53 signaling. In conclusion, 10 e, with prominent tumor selectivity and water solubility, could be a promising candidate for the treatment of cancer and, as such, warrants further investigation.

Efficacy of novel methylenecyclohexenone derivatives as TrxR inhibitors in suppressing the proliferation and metastasis of human cancer cells.

A series of mono- and di-methylenecyclohexenone derivatives, 3a-f and 4a-f, respectively, were designed and synthesized from piperlongumine (PL) and their in vitro and in vivo pharmacological properties were evaluated. A majority of the compounds exhibited a potent antiproliferative effect on five human cancer cell lines, especially those causing breast cancer. Compound 4f showed the highest antiproliferative potency among all of the compounds, almost a 10-fold higher inhibitory potency against thioredoxin reductase (TrxR) compared with PL in cells causing breast cancer. In addition, 4f was found to increase the levels of reactive oxygen species (ROS), thus leading to more potent antiproliferative effects. More importantly, the suppression assays of migration and invasion revealed that compound 4f could reverse the epithelial-mesenchymal transition induced by the transforming growth factor ß1, and exhibit prominent anti-metastasis effects. Compound 4f also showed strong inhibition potency toward solid tumors of breast cancer in vivo. Our findings show that compound 4f is a promising therapeutic candidate in the treatment of breast cancer, which, however, needs further research to be proved.

Novel Hybrid CHC from ß-carboline and N-Hydroxyacrylamide Overcomes Drug-Resistant Hepatocellular Carcinoma by Promoting Apoptosis, DNA Damage, and Cell Cycle Arrest.

A novel hybrid CHC was designed and synthesized by conjugating ß-carboline with an important active fragment N-hydroxyacrylamide of histone deacetylase (HDAC) inhibitor by an amide linkage to enhance antitumor efficacy/potency or even block drug resistance. CHC displayed high antiproliferative effects against drug-sensitive SUMM-7721, Bel7402, Huh7, and HCT116 cells and drug-resistant Bel7402/5FU cells with IC50 values ranging from 1.84 to 3.27 µM, which were two-to four-fold lower than those of FDA-approved HDAC inhibitor SAHA. However, CHC had relatively weak effect on non-tumor hepatic LO2 cells. Furthermore, CHC exhibited selective HDAC1/6 inhibitory effects and simultaneously augmented the acetylated histone H3/H4 and α-tubulin, which may make a great contribution to their antiproliferative effects. In addition, CHC also electrostatically interacted with CT-DNA, exerted remarkable cellular apoptosis by regulating the expression of apoptosis-related proteins and DNA damage proteins in Bel7402/5FU cells, and significantly accumulated cancer cells at the G2/M phase of the cell cycle by suppressing CDK1 and cyclin B protein with greater potency than SAHA-treated groups. Finally, CHC displayed strong inhibitory potency to drug-resistant hepatic tumors in mice. Our designed and synthetic hybrid CHC could be further developed as a significant and selective anticancer agent to potentially treat drug-resistant hepatocellular carcinoma.

Hydroxamic Acid Derivatives of ß-Carboline/Hydroxycinnamic Acid Hybrids Inducing Apoptosis and Autophagy through the PI3K/Akt/mTOR Pathways.

Naturally occurring ß-carbolines are known to have antitumor activities but with limited effectiveness. In order to improve their efficacy, a series of new hydroxamic-acid-containing ß-carbolines connected via a hydroxycinnamic acid moitey (12a-f) were developed to incorporate histone deacetylase (HDAC) inhibition for possible synergistic effects. When evaluated in in vitro assays, most of the analogues showed significant antitumor activities against four human cancer cells. In particular, 12b showed the highest cytotoxic potency of the series, including drug-resistant Bel7402 cells, but had minimal effect on normal hepatic LO2 cells. These compounds also showed excellent inhibitory effects against HDAC1/6, which appear to contribute greatly to their antiproliferative properties. Compound 12b enhanced the acetylation levels of histone H3 and α-tubulin and induced greater cancer cell apoptosis than the FDA-approved HDAC inhibitor SAHA by regulating expression of apoptotic proteins Bax, Bcl-2, and caspase 3. Importantly, 12b also induced a significant amount of autophagic flux activity in Bel7402 cells by increasing the expression of Beclin-1 and LC3-II proteins and decreasing that of LC3-I and p62. Finally, 12b significantly inhibited PI3K/Akt/mTOR signaling, an important cell-growth-promoting pathway aberrantly activated in many cancers. Together, the results suggest that these hydroxamic-acid-containing ß-carboline derivatives may be new leads for the discovery of agents for the treatment of human carcinoma cancers.

A novel harmine derivative, N-(4-(hydroxycarbamoyl)benzyl)-1-(4- methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (HBC), as histone deacetylase inhibitor: in vitro antiproliferation, apoptosis induction, cell cycle arrest, and antimetastatic effects.

This study aims to design and synthesize a novel harmine derivative N-(4-(hydroxycarbamoyl) benzyl)-1-(4-methoxyphenyl)-9H-pyrido [3,4-b]indole-3-carboxamide (HBC) as histone deacetylase (HDAC) inhibitor, and evaluate its antitumor activities and anti-metastasis mechanism. HBC not only exerted significant ant-proliferation activity against five human cancer cell lines, especially for HepG2 cell with an IC50 value of 2.21 µM, which is nearly three-fold lower than SAHA (IC50 = 6.26 µM), but also showed selective HDAC1/6 inhibitory effects in vitro. However, HBC had little effect on normal hepatic cells LO2. Furthermore, HBC simultaneously increased the acetylation of histone H3, H4, and α-tubulin, induced hypochromism by electrostatical interaction with CT-DNA, triggered more significant cancer cell apoptosis and cell cycle arrest at G2/M than SAHA by inhibition of both CDK1 and cyclin B in a concentration dependent manner. In addition, scratch and invasion assay showed that HBC also dose-dependently suppressed migration and invasion capacities of highly metastatic HCC HepG2 cells through down-regulated the expression of tumor metastasis related proteins MMP-2 and MMP-9, significantly better than SAHA. Finally, HBC showed low acute toxicity to mice and significant growth inhibition of the hepatoma tumor in vivo. These results demonstrate that novel harmine-based HDAC inhibitor HBC not only exhibited selective HDAC1/6 inhibitory activity and significant in vitro and in vivo antitumor activity, but also possessed DNA binding effect, apoptosis induction, cell cycle arrest effects, and potent anti-metastasis mechanisms, which may hold great promise as therapeutic agent targeting HDAC1/6 for the intervention of human cancers.

Development of novel ß-carboline-based hydroxamate derivatives as HDAC inhibitors with antiproliferative and antimetastatic activities in human cancer cells.

A series of novel ß-carboline-based hydroxamate derivatives 12a-k were designed and synthesized, and their biological activities in a series of in vitro assays were evaluated. Several of these ß-carboline derivatives not only showed excellent HDAC1/3/6 inhibitory effects, but also displayed significant antitumor activities against five human cancer cells. The most potent compound 12f demonstrated the highest anticancer potency against cancer cell lines with IC50 values of 0.53-1.56 µM, which was considerably more potent than harmine (IC50 = 46.7-55.3 µM) and also three-to ten-fold lower than that of SAHA (IC50 = 4.48-6.26 µM). Immunoblot analysis revealed that 12f dose-dependently inhibited histone H3 and α-tubulin acetylation, confirming its HDAC inhibitory effects. Moreover, 12f significantly arrested HepG2 cells at G2/M phase through inhibiting cell cycle related protein CDK1 and cyclin B in a concentration dependent manner. Interestingly, 12f also exerted strong anti-metastasis activity by simultaneously reducing the protein level of MMP2 and MMP9 and inhibiting MAPK signaling pathway.

Development of novel ß-carboline-based hydroxamate derivatives as HDAC inhibitors with DNA damage and apoptosis inducing abilities.

A series of novel ß-carboline-based hydroxamate derivatives (8a-n) as HDAC inhibitors have been designed and synthesized. Most of these compounds displayed potent histone deacetylase inhibitory effects and good antiproliferative activity with IC50s in the low micromolar range. One of the most potent compounds (8k) showed the strongest inhibition of the proliferation of human hepatocellular carcinoma (HCC) cells in vitro, with IC50 values lower than that of the currently approved HDAC inhibitor SAHA. Compound 8k also increased acetylation of histone H3 and α-tubulin, consistent with its potent HDAC inhibition. Importantly, 8k induced hypochromism by electrostatic interactions with CT-DNA, suggesting potential induction of DNA damage. Finally, 8k significantly induced HepG2 cell apoptosis by regulating apoptotic relative proteins expression. Together, our findings suggest that these novel ß-carboline-based hydroxamate derivatives may provide a new framework for the discovery of novel antitumor agents for the intervention of human carcinoma cells.

Design, synthesis and biological evaluation of hybrids of ß-carboline and salicylic acid as potential anticancer and apoptosis inducing agents.

A novel series of hybrids (7a-l, 8a-l) from ß-carboline and salicylic acid (SA) were designed and synthesized, and their in vitro biological activities were evaluated. Most of the hybrids displayed potent antiproliferative activity against five cancer cell lines in vitro, showing potencies superior to 5-FU and harmine. In particular, compound 8h selectively inhibited proliferation of liver cancer SMMC-7721 cells but not normal liver LO2 cells, and displayed greater inhibitory selectivity than intermediate 5h and SA. 8h also induced cancer cell apoptosis in an Annexin V-FITC/propidium iodide flow cytometry assay, and triggered the mitochondrial/caspase apoptosis by decreasing mitochondrial membrane potential which was associated with up-regulation of Bax, down-regulation of Bcl-2 and activation levels of the caspase cascade in a concentration-dependent manner. Our findings suggest that the ß-carboline/SA hybrids may hold greater promise as therapeutic agents for the intervention of human cancers.