The Role Of Notch Signaling In Multiple Myeloma And Involved Mechanism

Multiple myeloma, which occurs in the elderly, is a malignancy of hematologicalsystem. Due to lack of effective treatments, it is recognized as an incurable disease.Conventional chemotherapy did not acquire good effects, because it made the patientssuffer a lot and had a very high rate of resistance. Hematological system stem celltransplantation (HSCT) costs much, and the selective donors are limited. The risk oftransplantation is associated with aging. In recent years, the appearance of proteasomeinhibitors, such as bortezomib and carfilzomib, had an epoch-making significance in thetreatment of MM. But most multiple myeloma patients came into drug-resistanceultimately. So, it is necessary to further study the pathogenesis of MM and look for safeand effective anti-tumor drugs or chemosensitive medicine.Curcumin is an active constituent extracted from the rhizome of zingiberaceaeincluding turmeric, zedoary and radix curcuma. It has wide pharmacologic effects, such asanti-inflammatory, antioxidation, anticoagulation, hypolipidemic, antiatherosclerotic,antirheumatoid, anticancer, anti-pathogeny microorganism and neuroprotection. Recently,the anticancer of curcumin appeals to us. Some studies show that curcumin has inhibitoryeffect on many cancers. Curcumin can also increase the sensivity of cancer cells tochemotherapy and radiation, but it has chemprotective and radioprotective effects onnormal cells.Notch signaling plays an important role in the proliferation and apoptosis ofhematopoietic cells. Notch1is one of the four single-pass transmembrane cell surfacereceptors (Notch1-4in mammals). Recently, it is reported that notch1expressed heavilyon myeloma cells. It suppressed apoptosis of myeloma cells and resulted in chemoresistance. This data support that notch1may be a target for the treatment ofmyeloma. In this study, we explore the chemosesitization by curcumin in MM cells，andthe role of notch1signaling involved in it.Objective: The present study is designed to investigate the effect of curcumin on thechemosesitization to bortezomib in MM cells, and the role of notch1signaling involved init.Methods:（1）RPMI8226and KAS-6/1were treated with different concentration ofcurcumin. After different times, the cell proliferation was detected by MTT assay; the cellapoptosis was detected by Annexin-V/PI with flow cytometry; the protein expression ofcleaved notch1and apoptosis-related protein cleaved caspase3were detected by westernblotting.（2）RPMI8226, OPM2, RPMI8226-bortezomib resistant（RPMI8226-V5R）,OPM2-bortezomib resistan（tOPM2-V5R） and CD138(+) cells from patients were treatedwith curcumin or bortezomib, or combination of the two. The cell proliferation wasdetected by MTT assay; the cell apoptosis was detected by Annexin-V/PI with flowcytometry; the protein expression of cleaved notch1and cleaved caspase3were detected byusing western blotting.（3） RPMI8226, OPM2, RPMI8226-bortezomib resistant,OPM2-bortezomib resistant and CD138(+) cells from patients were treated with DAPT orbortezomib, or combination of the two. The cell proliferation was detected by MTT assay;the cell apoptosis was detected by Annexin-V/PI with flow cytometry; the proteinexpression of cleaved notch1and cleaved caspase3were detected by using western blotting.（4）RPMI8226-V5R and OPM2-V5R were transfected with Notch1-siRNA andNT-siRNA(as control), then were treated with bortezomib. The cell proliferation wasdetected by MTT assay. The protein expression of cleaved notch1and cleaved caspase3were detected by using western blotting.Results：（1）Curcumin can inhibit the proliferation and increase the apoptosis ofRPMI8226and KAS-6/1in a dose dependant manner; the protein expression of cleavednotch1decreasd, while the expression of apoptosis-related protein cleaved caspase3increased with the augment of curcumin concentration.（2）In all the four cell lines andCD138(+) cells from patients, the proliferation of experimental groups were better thanthat of the control group; the the proliferation of combined drug group was lower than thesimple drug groups. Curcumin and bortezomib can induce the apoptosis of RPMI8226and OPM2, and the combination of them can make a greater apoptosis. Curcumin, not only canpromote the inhibitory effect of bortezomib on the expression of cleaved notch1, but alsocan facilitate the promoting effect of bortezomib on the expression of cleaved caspase3.(3)In all the four cell lines and CD138(+) cells from patients, the proliferation ofexperimental groups were better than that of the control group; the the proliferation ofcombined drug group was lower than the simple drug groups. DAPT, not only can promotethe inhibitory effect of bortezomib on the expression of cleaved notch1, but also canfacilitate the promoting effect of bortezomib on the expression of cleaved caspase3.(4) Inthe RPMI8226-V5R and OPM2-V5R cells，the inhibitive expression of cleaved notch1bytransfecting Notch1-siRNA can promote the inhibitory effect of bortezomib on theproliferation of cells，and significantly increases the expression of cleaved caspase3. Whilethe NT-siRNA transfected groups do not have this effect.Conclution: Curcumin can increase the chemosesitization to bortezomib in MM cellsvia the inhibition of notch1signaling. Multiple myeloma is a plasma cell malignancy presented with the development ofosteolytic lesions and/or diffuse osteopenia which manifested with bone pain andpathological fractures. The pathogenesis of myeloma bone disease is reported to be theunbalanced bone remodeling with increased osteoclastogenesis and decreased osteogenesis.Increased RANKL/OPG ration in MSCs from myeloma patients is widely accepted as themechanism of osteoclasts activation and bone resorption, while the reason for osteogenesissuppression is far from elucidated.The most important property of MSCs is the multipotent differentiation ability, one ofwhich is osteogenesis. Recent study reveals that MSCs derived from patients with multiplemyeloma show significant defects compared with MSCs from normal donors, including the impaired osteogenic differentiation ability. The blockade of osteogenic differentiation ofmesenchymal progenitors to mature osteoblasts is partialy responsible for the impairmentof osteoblast activity in MM. It is reported that over-production of soluble inhibitors,interleukin-3, secreted Frizzled-related protein-2(sFR-2), tumor necrosis factor (TNF) anddickkopf-1(Dkk-1) by myeloma cells and stomal cells may impede osteoblasts formationand maturation.Notch signaling is an evolutionally conserved pathway system. It controls various celleffects including proliferation, differentiation, and cell-fate decisions. Notch1is one of thefour single-pass transmembrane cell surface receptors (Notch1–4in mammals). Uponbinding of its ligands, notch1undergoes proteolytic cleavages and releases its cleaved parts,the Notch intracellular domain (NICD), which translocates to the nucleus and interactswith a transcription factor to activate transcription of target genes. It is reported that notch1signaling inhibited osteogenic differentiation potential and maintain the stem cell propertyof mesenchymal progenitors.Bortezomib is the first FDA approved proteasome inhibitor. It has demonstratedefficacy in the treatment of both newly diagnosed and relapsed/refractory myeloma. It isreported that bortezomib not only inhibited osteoclast function but also enhancedosteoblast activity both in vitro and in vivo. CFZ, the second generation of proteasomeinhibitor, has exhibited potent anti-myeloma efficacy and decreased side effects comparedwith bortezomib. Beside the anti-myeloma efficacy, CZF has the ability to enhanceosteoblast formation and function. It is reported to promote osteogenic differentiation andmatrix mineralization in MSCs. However, the exact mechanism of osteogesis promotioneffect of CFZ is still far from elucidated. In this study, we detected the effect of CZF on theostegenic differentiation potential of MSCs from myeloma patients and involvedmechanism.Objective: The present study is designed to investigate the osteogenic differentiationpotential and the activity of notch1of MM-MSCs,and to explore the relationship betweennotch1signaling and osteogenic differentiation potential.We also want to detecte the effectof CZF on the ostegenic differentiation potential of MSCs from myeloma patients andinvolved mechanism.Methods:(1) MM-MSCs and ND-MSCs (as control) were cultured in osteogenicmedium for two weeks. Both the mRNA expression and protein expression of osteogenic markers Runx2, OCN and Osx were detected by using real-time RT-PCR and westernblotting. Calcium deposit was detected with von Kossa staining. The mRNA expression ofHes1(a downstream regulator of notch1) before and after oseogenic induction wasdetected by using real-time RT-PCR. The protein expression of Hes1and cleaved notch1before and after oseogenic induction were detected by using western blotting.（2）MM-MSCs were cultured in the osteogenic induction medium containing Notch1inhibitorDAPT (5nM) or Vehicle (control) for two weeks. Real-time RT-PCR was used to detect themRNA expression of Hes1and osteogenic differentiation markers Runx2, OCN and Osx.The protein expression of Runx2, OCN, Osx, cleaved notch1and Hes1were detected byusing western blotting. Calcium deposit was detected with von kossa staining.（3）MM-MSCs was cultured in the medium containing CFZ (0,2.5nM and5.0nM) for twoweeks. Real-time RT-PCR was used to detect the mRNA expression of Hes1andosteogenic differentiation markers Runx2, OCN and Osx. The protein expression of Runx2,OCN, Osx, cleaved notch1and Hes1were detected by using western blotting. Calciumdeposit was detected with von kossa staining.Results：（1）Both the mRNA expression and protein expression of osteogenic markersRunx2, OCN and Osx were decreased in MM-MSCs compared with those in ND-MSCs.Calcium deposit of MM-MSCs was weakened. The mRNA expression of Hes1wasinhibited after oseogenic induction in ND-MSCs, while it remained relatively stable inMM-MSCs before and after OI. Protein expressions of both cleaved notch1and Hes1decreased in ND-MSCs after OI, but not in MM-MSCs.（2）DAPT inhibited the mRNAexpression of Hes1and the protein expression of cleaved notch1and Hes1. The mRNAand protein expression of osteogenic markers, Runx2, OCN and Osx, were all promoted toa higher level. Calcium deposit of MM-MSCs was increased（.3）CFZ inhibited the mRNAexpression of Hes1in a dose-dependent manner. The protein expression of cleaved notch1and Hes1both decreased after CFZ treatment. The osteogenic differentiation ability waspromoted by CFZ in a dose-dependent manner, which was demonstrated by elevatedosteogenic differentiation markers, Runx2, Osx and OCN and more calcium deposit.Conclution: Accompanied by impairment of notch1deactivation, the osteogenicdifferentiation potential of MM-MSCs was decresed. CFZ treatment promoted ostegenicdifferentiation potential of MSCs via notch1signaling pathway inhbition.