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.

[Study on effect of different processing methods on chemical components of Chuanxiong Rhizoma].

To determine the content of extracts in different processed products of Chuanxiong Rhizoma and the content of chlorogenic acid, ferulic acid, senkyunolide Ⅰ, coniferyl ferulate, senkyunolide A and ligustilide, in order to study the effect of different proces-sing methods on the alcohol-soluble extract and the content of six ingredients of Chuanxiong Rhizoma. The extract was determined according to the alcohol-soluble extract determination method set forth in item 2201 of the 2020 Chinese Pharmacopoeia Ⅳ; the content was determined by using Agilent TC-C_(18) column(4.6 mm×250 mm, 5 µm) for gradient elution, with acetonitrile(A)-0.5% acetic acid solution(B) as the mobile phase; the column temperature was at 30 ℃; the flow rate was 1.0 mL·min~(-1), the detection wavelength was 285 nm; and the injection volume was 10 µL. Compared with Chuanxiong Rhizoma, the extracts of processed products all increased significantly; by the degree of increase, the order was stir-frying Chuanxiong Rhizoma with honey>stir-frying Chuanxiong Rhizoma with rice wine>stir-frying Chuanxiong Rhizoma with Angelicae Dahuricae Radix decoction>stir-frying Chuanxiong Rhizoma with tea decoction; the HPLC method was convenient and reliable, with a high linear relationship of chlorogenic acid, ferulic acid, senkyunolide Ⅰ, coniferyl ferulate, senkyunolide A and ligustilide, and a high precision, repeatability, stability and the sample recovery rate in Chuanxiong Rhizoma and its processed products. There were 15 chromatographic peaks before and after processing, eight of them were identified. Compared with the pre-processing, two chromatographic peaks were added after the stir-frying with honey and rice wine; and four chromatographic peaks were added after the processing with Angelicae Dahuricae Radix decoction; the contents of chlorogenic acid, ferulic acid, senkyunolide Ⅰ, coniferyl ferulate, senkyunolide A, and ligustilide in stir-frying Chuanxiong Rhizoma with rice wine were all reduced. Except for the content of ferulic acid that increased, the content of the other five components decreased in stir-frying Chuanxiong Rhizoma with honey, stir-frying Chuanxiong Rhizoma with tea decoction, and stir-frying Chuanxiong Rhizoma with Angelicae Dahuricae Radix decoction. Rice wine, honey, decoction of tea and Angelicae Dahuricae Radix could all promote the dissolution of chemical components in Chuanxiong Rhizoma, and increase the content of extract; the changes in the contents of six components of different processed products could provide a certain basis for studying chemical composition and efficacy of different processed products of Chuanxiong Rhizoma.