MAPK Cascade Signaling Is Involved in α-MMC Induced Growth Inhibition of Multiple Myeloma MM.1S Cells via G2 Arrest and Mitochondrial-Pathway-Dependent Apoptosis In Vitro.

Multiple myeloma is a hematological malignancy characterized by the unrestricted proliferation of plasma cells that secrete monoclonal immunoglobulins in the bone marrow. Alpha-momorcharin (α-MMC) is a type I ribosome-inactivating protein extracted from the seeds of the edible plant Momordica charantia L., which has a variety of biological activities. This study aimed to investigate the inhibitory effect of α-MMC on the proliferation of multiple myeloma MM.1S cells and the molecular mechanism of MM.1S cell death induced through the activation of cell signal transduction pathways. The cell counting kit-8 (CCK-8) assay was used to determine the inhibitory effect of α-MMC on the proliferation of MM.1S cells and its toxic effect on normal human peripheral blood mononuclear cells (PBMCs). The effect of α-MMC on the MM.1S cells' morphology was observed via inverted microscope imaging. The effects of α-MMC on the MM.1S cell cycle, mitochondrial membrane potential (MMP), and apoptosis were explored using propidium iodide, JC-1, annexin V- fluorescein isothiocyanate/propidium iodide fluorescence staining, and flow cytometry (FCM) analysis. Western blot was used to detect the expressions levels of apoptosis-related proteins and MAPK-signaling-pathway-related proteins in MM.1S cells induced by α-MMC. The results of the CCK-8 showed that in the concentration range of no significant toxicity to PBMCs, α-MMC inhibited the proliferation of MM.1S cells in a time-dependent and concentration-dependent manner, and the IC50 value was 13.04 and 7.518 µg/mL for 24 and 48 h, respectively. Through inverted microscope imaging, it was observed that α-MMC induced a typical apoptotic morphology in MM.1S cells. The results of the FCM detection and analysis showed that α-MMC could arrest the MM.1S cells cycle at the G2 phase, decrease the MMP, and induce cell apoptosis. Western blot analysis found that α-MMC upregulated the expression levels of Bax, Bid, cleaved caspase-3, and cleaved PARP, and downregulated the expression levels of Mcl-1. At the same time, α-MMC decreased the expression levels of p-c-Raf, p-MEK1/2, p-ERK1/2, p-MSK1, and p-P90RSK, and increased the expression levels of p-p38, p-SPAK/JNK, p-c-Jun, and p-ATF2. The above results suggest that α-MMC can inhibit the proliferation of multiple myeloma MM.1S cells. MAPK cascade signaling is involved in the growth inhibition effect of α-MMC on MM.1S cells via cycle arrest and mitochondrial-pathway-dependent apoptosis.

Identification and transcriptional activity analysis of core regulatory region of human guanylate binding protein 5 gene promoter / 中国生物制品学杂志

@#Objective To construct luciferase reporter plasmids of truncated fragments of different lengths of human guanylate binding protein 5(GBP5)gene promoter and analyze the transcriptional activity of each fragment to determine the core regulatory region.Methods GBP5promoter sequence was amplified by PCR,truncated into five fragments of different lengths and connected to pGL3-basic plasmid.The constructed recombinant plasmids pGL3-GBP5-11/21/31/41/51were transfected into 293FT cells and detected for luciferase activity.The binding sites of transcription factors in GBP5promoter region were predicted by JASPAR software,and Yin-Yang transcription factor 1(YY1)targeting the core regulatory region was selected and verified for the transcriptional regulatory activity.The CDS sequence of YY1 was amplified by PCR to construct the overexpression plasmid pIRES2-EGFP-YY1,which was then co-transfected to 293FT cells with plasmids pGL3-GBP5-21(-1 623 ～ +47 bp)and internal reference plasmid pRL-CMV,and detected for luciferase activity to analyze the regulation of transcription factor YY1 on GBP5 promoter activity.Results Colony PCR and double enzyme digestion identification proved that the plasmid of human GBP5 promoter reporter gene was correctly constructed;JASPAR software predicted that there were multiple transcription factor binding sites such as STAT1,YY1 and Foxp3 in GBP5promoter region.Double luciferase activity assay showed that pGL3-GBP5-21(-1 623 ～ +47 bp)showed the highest promoter activity,while the promoter activity of pGL3-GBP5-41(-520 ～ +47 bp)decreased significantly,suggesting that the core region of GBP5 promoter was located at upstream-1 623 ～-520 bp of 5 'UTR;Overexpression of YY1 significantly activated the GBP5 promoter activity and regulated the expression of GBP5.Conclusion The core regulatory region of human GBP5 promoter was located in upstream-1 623 ～-520 bp of the 5 'UTR,with a binding site of transcription factor YY1 existing in this region.Meanwhile,overexpression of YY1 significantly effected the activity of GBP5 promoter.

Cdc25C/cdc2/cyclin B, raf/MEK/ERK and PERK/eIF2α/CHOP pathways are involved in forskolin-induced growth inhibition of MM.1S cells by G2/M arrest and mitochondrion-dependent apoptosis.

Multiple myeloma (MM) remains an incurable hematological malignancy characterized by proliferation and accumulation of plasma cells in the bone marrow. Innovative and effective therapeutic approaches that are able to improve the outcome and the survival of MM sufferers, especially the identification of novel natural compounds and investigation of their anti-MM mechanisms, are needed. Here, we investigated the effects and the potential mechanisms against MM of forskolin, a diterpene derived from the medicinal plant Coleus forskohlii, in MM cell line MM.1S. CCK-8 assay showed that forskolin significantly inhibited MM.1S cells viability in a time- and dose-dependent manner. Furthermore, we demonstrated that forskolin induced G2/M phase arrest with a remarkable increase of p-cdc25c, p-cdc2, and a decrease of cyclin B1, indicating the suppression of cdc25C/cdc2/cyclin B pathway. Moreover, we found that forskolin induced mitochondrion-dependent apoptosis which was accompanied by the increase of pro-apoptotic proteins Bax, Bad, Bim and Bid, the decrease of anti-apoptotic proteins Bcl-2 and Bcl-xl, the changes of the mitochondrial membrane potential (MMP) and increase of cleaved caspase-9, cleaved caspase-3 and cleaved PARP. Of note, we demonstrated that forskolin induced a decrease of p-C-Raf, p-MEK, p-ERK1/2 and p-p90Rsk, and an increase of p-PERK, p-eIF2α and CHOP, which indicated that the inhibition of Raf/MEK/ERK pathway and activation of PERK/eIF2α/CHOP pathway were involved, at least partially, in forskolin-induced MM.1S cells apoptosis. These findings confirm the anti-MM action of forskolin and extend the understanding of its anti-MM mechanism in MM.1S cells, as well as reinforcing the evidence for forskolin as a natural chemotherapeutic compound against MM.

Lycorine hydrochloride induces reactive oxygen species-mediated apoptosis via the mitochondrial apoptotic pathway and the JNK signaling pathway in the oral squamous cell carcinoma HSC-3 cell line.

Poor drug efficacy is a prominent cause of oral squamous cell carcinoma (OSCC) treatment failure. Although increased efforts in developing OSCC therapeutic strategies have been achieved in recent decades, the 5-year survival rate of patients with OSCC remains poor and effective drugs to treat OSCC are lacking. The aim of the present study was to investigate the apoptotic effect caused by lycorine hydrochloride (LH) and to identify its mechanism in the OSCC HSC-3 cell line. The findings demonstrated that LH effectively induced HSC-3 cell apoptosis and cell cycle arrest at the G0/G1 phase, resulting in the inhibition of cell proliferation. Furthermore, it was found that LH increased reactive oxygen species (ROS) production, triggered mitochondrial membrane potential (MMP) disorder, enhanced the protein expression levels of Bax, Bim, cleaved caspase-9, caspase-3 and poly(ADP-ribose) polymerase 1 and decreased Mcl-1 expression. The protein expression levels of important members of the JNK signaling pathway, including phosphorylated (p)-JNK, p-mitogen-activated protein kinase kinase 4 and p-c-Jun, were significantly increased in LH-treated cells, accompanied by an increase in ROS. However, N-acetyl cysteine (NAC), a potent antioxidant, reversed the upregulated mRNA expression of c-Jun, as well as the enhanced ROS production, the disorder of MMP and the apoptosis of HSC-3 cells induced by LH. These results suggested that LH may induce HSC-3 cell apoptosis via the ROS-mediated mitochondrial apoptotic pathway and the JNK signaling pathway, which indicated that LH may be a potential drug candidate for anti-OSCC therapy.