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May 11, 2009 - tion kit according to the manufacturer's instructions. Briefly, KM3 cells (5 Â 105) .... comitant decrease in the pro-form (32 kDa). Similarly, a.
Acta Biochim Biophys Sin (2009): 1018 – 1026 | ª The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmp094. Advance Access Publication 10 November 2009

Potentiation of (2)-epigallocatechin-3-gallate-induced apoptosis by bortezomib in multiple myeloma cells Qing Wang, Juan Li*, Jingli Gu, Beihui Huang, Ying Zhao, Dong Zheng, Yan Ding, and Lijin Zeng Department of Hematology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China *Correspondence address. Tel: þ86-20-87755766-8831; Fax: þ86-20-87333455; E-mail: [email protected]

The green tea constituent, (2)-epigallocatechin-3gallate (EGCG), has chemopreventive and anticancer effects. This is partially because of the selective ability of EGCG to induce apoptosis and death in cancer cells without affecting normal cells. In the present study, the activity of EGCG against the myeloma cell line, KM3, was examined. Our results demonstrated, for the first time, that the treatment of the KM3 cell line with EGCG inhibits cell proliferation and induces apoptosis, and there is a synergistic effect when EGCG and bortezomib are combined. Further experiments showed that this effect involves the NF-kB pathway. EGCG inhibits the expression of the P65 mRNA and P65/pP65 protein, meanwhile it downregulates pIkBa expression and upregulates IkBa expression. EGCG also activates caspase-3, -8, cleaved caspase-9, and poly-ADP-ribose polymerase (PARP) and subsequent apoptosis. These findings provided experimental evidence for efficacy of EGCG alone or in combination with bortezomib in multiple myeloma therapy.

Keywords (2)-epigallocatechin-3-gallate; apoptosis; NF-kB; bortezomib; multiple myeloma Received: May 11, 2009

Accepted: August 7, 2009

Introduction Despite advances in systemic and supportive therapies, multiple myeloma (MM) remains an incurable plasma cell malignancy due to both intrinsic and acquired drug resistance [1,2]. Thus, there is a need to find new ways to increase the efficacy of MM therapy. (2)-Epigallocatechin-3-gallate (EGCG) has received much attention over the last few years as a potential cancer chemopreventive and chemotherapeutic agent, possibly because of its wide range of effects on a Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 1018

number of cellular processes and its efficacy in many tumor model systems [3,4]. The mechanism underlying EGCG-mediated apoptosis of cancer cells is incompletely understood. Such an understanding, however, could prove the importance of developing EGCG and related compounds as single agents or in combination with other drugs for the prevention and/or therapy of cancer. The transcription factor, NF-kB, plays a critical role in the regulation of genes related to cell survival, proliferation, and apoptosis [5,6]. Under normal conditions, NF-kB is present in the cytoplasm as an inactive heterotrimer consisting of P50, P65, and IkBa subunits. Upon activation, IkBa undergoes phosphorylation and ubiquitination-dependent degradation by the 26S proteosome, and thus the P65 subunit of the NF-kB containing transactivation domain is translocated to the nucleus and bound to a specific consensus sequence in the DNA. The binding activates diverse gene expression, which in turn results in gene transcription [7]. In recent years, considerable attention has been focused on NF-kB inhibition as a target for developing agents for cancer prevention and therapy [8,9]. Studies have shown that EGCG is capable of inhibiting NF-kB activation and inducing apoptosis in cancer cells [10–12]. There are many other putative mechanisms for EGCG in cancer therapy. EGCG can suppress the activity of MMP-9 in PC-3 cells in the presence of Zn2þ; as a result, the ability of cells to migrate is significantly decreased [13]. In human hepatocellular carcinoma cells, EGCG can downregulate COX-2 and Bcl-2, and consequently activating caspase-9 and -3 [14]. EGCG-induced apoptosis involves both the extracellular signal-regulated protein kinase and c-jun N-terminal kinase pathways in Jurkat cells [15]. In LNCaP prostate cancer cells, EGCG suppresses the expression of androgen receptor signaling and prostatespecific antigen in different stages of progression [16]. Using L-929 cells, Han et al. [17] reported that the

Potentiation of EGCG-induced apoptosis by bortezomib in multiple myeloma cells

cellular sensitivity and response to EGCG is incorporated into the cytoplasm of cells and further translocated into the nucleus in a time-dependent manner. EGCG also acts as a topoisomerase I inhibitor in human colon cancer cells [18]. In human ovarian carcinoma SKOV3 cells, EGCG which inhibits ligand-induced AhRE (aryl hydrocarbon receptor element) binding and AhR (aryl hydrocarbon receptor)-mediated transcriptional activity decreased the levels of several cancer-related hsp90 client proteins, such as ErbB2, Raf-1, and phospho-AKT. EGCG also modifies the association of hsp90 with several chaperones. These data indicate that EGCG is a novel hsp90 inhibitor [19]. In addition to these pathways, lysosomes involved as the primary organelles in apoptotic cell death are induced by some signals, such as B cell receptor-induced apoptosis [20]. There have been studies demonstrating the role of EGCG in disruption of the mitochondrial pathway and induction of apoptosis [21]. However, little data regarding EGCG exist as it pertains to MM. Herein, we demonstrate the essential role of caspases with EGCG alone, or in association with bortezomib, in mediating inhibition of NF-kB and induction of apoptosis in myeloma KM3 cells [22]. The results showed that the mechanism responsible for EGCG-mediated inhibition of NF-kB is via NF-kB/P65 and phosphorylation and degradation of IkBa. EGCG also activates caspasemediated induction of apoptosis, and there is a synergistic effect of the combination of EGCG and bortezomib.

Materials and Methods Cell lines and reagents The KM3 cell line was kindly provided by Prof. Jian Hou (Second Military Medical University, Shanghai, China). The cell line was cultured in 10% fetal bovine serum/RPMI 1640 supplemented with antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) in a humidified atmosphere with 5% CO2 at 378C. EGCG was purchased from Sigma-Aldrich (St. Louis, USA). The proteasome inhibitor, bortezomib (PS-341, Velcade), was purchased from the Millennium Predictive Medicine, Inc. (Cambridge, USA). Antibody against P65, phosphorylated P65(Ser536) ( pP65), IkBa, phosphorylated IkBa(Ser32/36) ( pIkBa), caspase-3, -8, cleaved caspase-9, poly-ADP-ribose polymerase (PARP), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, USA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse and FITC-conjugated

goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Annexin V-FITC and a propidium iodide (PI) kit (Oncogene Research Products, San Diego, USA) and a PrimeScriptTM 1st strand cDNA synthesis kit and SYBRw Premix Ex TaqTM II (Perfect Real Time; TaKaRa, Dalian, China) were used in experiments as indicated.

Treatment and growth of cells EGCG was dissolved in PBS ( pH 7.4) and used for the treatment of cells. The cells were treated with EGCG at 0, 25, 50, and 100 mM for 48 h in the dose-dependence assay and with or without bortezomib (20 nM) in the combination assay. For the time-dependence assay, the cells were treated with 50 mM EGCG for 0, 12, 24, and 48 h. Cells (5  105) were plated in 100-mm dishes and treated with EGCG, with or without bortezomib, and the number of viable cells was determined by Trypan blue exclusion in quintuplicate. Apoptosis assay A flow cytometry analysis of Annexin V-FITC- and PI-stained cells was performed using the apoptosis detection kit according to the manufacturer’s instructions. Briefly, KM3 cells (5  105) treated with EGCG with or without bortezomib were washed with 50 mM cold phosphate buffer ( pH 7.6) and centrifuged at 12,000 g for 5 min. The cells were suspended in 100 ml of binding buffer. A mixture of 5 ml of fluorescence-conjugated Annexin V (a Ca2þ-dependent and phospholipid-binding protein) and 2 ml of PI was added to the cell suspension and then incubated for 15 min at room temperature. The samples were analyzed for Annexin V binding within 1 h by flow cytometry. Comparative experiments were performed at the same time and analyzed with Cell Quest software. Data were obtained from a cell population from which debris was gated out. RNA extraction, quantification, and amplification RNA was extracted from cells after treatment with EGCG alone or in combination with bortezomib using commercially available kits according to the manufacturer’s instructions and quantified using a Nanodrop spectrophotometer (Labtech, East Sussex, UK). Complementary DNA (cDNA) was synthesized from equal quantities of RNA (1 mg) using the PrimeScriptTM 1st strand cDNA synthesis kit. Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 1019

Potentiation of EGCG-induced apoptosis by bortezomib in multiple myeloma cells

Real-time polymerase chain reaction A SYBR Green real-time polymerase chain reaction (PCR) was performed on cDNA extracted from cells after treatment with EGCG alone or in combination with bortezomib. Primer sequences were designed for P65 as follows: forward: 50 -CAACAACCCCTTCCAAGTTCC30 ; and reverse: 50 -TTCACTCGGCAGATCTTGAGC-30 . Amplification was done with an initial incubation at 958C for 2 min, followed by 958C for 30 s, 608C for 30 s, and 728C for 30 s, for a total of 40 cycles. Amplification of 18S rRNA was performed in the same reaction tubes as the internal standard (Applied Biosystems, Carlsbad, USA). Data analysis was completed using the 7500 Sequence Detection software (Applied Biosystems) and the 22DDCT method for relative quantitation.

Western blotting Protein extraction and immunoblotting analysis following the treatment of cells were performed as described previously [23]. Briefly, the media were aspirated, the cells were washed with cold PBS ( pH 7.4), and lysed by incubation with ice-cold lysis buffer (50 mM Tris –HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP-40, 1% Triton X-100, and 1 mM PMSF, pH 7.4) with freshly protease inhibitor cocktail (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, USA) over ice for 30 min. The cells were scraped and the lysate was collected in a microfuge tube and cleared by centrifugation at 12,000 g for 10 min at 48C; the supernatant (total cell lysate) was used or immediately stored at 2808C. The protein concentration was determined by DC Bio-Rad assay using the manufacturer’s protocol (Bio-Rad Laboratories, Hercules, USA). For immunoblotting analysis, 25–50 mg protein was suspended in Laemmli sample buffer (100 mM Tris–HCl, pH 6.8, 1% sodium dodecyl sulfate (SDS), 0.05% mercaptoethanol, 10% glycerol, and 0.001% bromophenol blue), boiled for 5 min, and electrophoresed on a 4–10% glycerol gradient SDS– polyacrylamide gel for 40 min at 60 V and 40 min at 170 V. Gels were electroblotted onto PVDF membranes at 100 V for 2.5 h in a Tris–glycine buffer system. Incubation with the indicated antibodies against either P65 (1:1000), pP65 (1:1000), IkBa (1:1000), pIkBa (1:1000), caspase-3 (1:1000), caspase-8 (1:1000), cleaved caspase-9 (1:1000), or PARP (1:1000) was performed at 48C overnight in TBS–Tween 20 (TBST) containing 1% BSA. Blots were washed with TBST and Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 1020

incubated in either horseradish peroxidase-conjugated anti-rabbit (1:2000) or anti-mouse (1:2000) antibody for 1 h in TBST containing 5% non-fat dry milk. After washing, specific proteins were detected using enhanced chemiluminescence, according to the instructions provided in the manual (Cell Signal Technology). For equal loading of protein, the membrane was stripped and re-probed with an anti-GAPDH antibody (1:1000).

Statistical analysis The data were expressed as mean + SD. Statistical analysis was performed using SPSS 13.0 software with Student’s t-test and one-way analysis of variance (ANOVA), followed by LSD significant difference test. A value of P , 0.05 (two-tailed test) was considered statistically significant. Analysis of synergism was performed according to median dose –effect analysis [24] using a commercially available software program (Calcusyn; Biosoft, Ferguson, USA).

Results EGCG-mediated growth inhibition and apoptosis in KM3 cells Using KM3 cells, we first evaluated the effect of EGCG on cell growth by Trypan blue exclusion assay. EGCG treatment (0, 25, 50, and 100 mM for 48 h) of KM3 cells resulted in a dose-dependent inhibition of cell growth. There was a significant difference between the 25, 50, and 100 mM and the control group (0 mM) (P , 0.01) [Fig. 1(A)]. In the time-dependence study, 50 mM EGCG resulted in cell inhibitory response, which was significant as early as 12 h post-EGCG treatment, with an increasing trend up to 48 h, as shown by Trypan blue exclusion assay. There was a significant difference between the 12, 24, and 48 h treatment groups and the control group (0 h) (P , 0.05) [Fig. 1(B)]. The apoptosis rate was observed by flow cytometry with Annexin V and PI kit after EGCG treatment (0, 25, 50, and 100 mM) for 48 h. The percentages of apoptosis were 8.5%, 12.1%, 29.5%, and 33.4% in the 0, 25, 50, and 100 mM groups, respectively. The P-values were all ,0.01 in the treatment groups compared with the control group (0 mM) [Fig. 1(C)]. Similarly, in the timedependence study, the percentages of apoptosis were 5.8%, 11.9%, 14.9%, and 29.5% in the 0, 12, 24, and 48 h groups, respectively. There was a significant difference (P , 0.01) between the other three groups and the control group (0 h) [Fig. 1(D)].

Potentiation of EGCG-induced apoptosis by bortezomib in multiple myeloma cells

Figure 1 EGCG inhibits cell growth and induces apoptosis in KM3 cells (A) Cells were incubated with different doses of EGCG for 48 h, as indicated. Cell growth inhibition was assessed by Trypan blue exclusion assay. Data are expressed as mean + SD from three individual experiments (**P , 0.01 compared with the control group). (B) Cells were incubated with EGCG (50 mM) for different times, as indicated. Cell growth inhibition was assessed by Trypan blue exclusion assay. The data shown are the mean + SD from three individual experiments (*P , 0.05 compared with the control group). (C) Cells were treated as in (A). Apoptosis was evaluated by Annexin V staining and FACScan analysis. The LR quadrant indicates the percentage of early apoptotic cells. (D) Cells were treated as in (B). Apoptosis was evaluated by Annexin V staining and FACScan analysis. The LR quadrant indicates the percentage of early apoptotic cells.

EGCG inhibits NF-kB/P65 expression, downregulates pIkBa, and upregulates IkBa in KM3 cells Next, we investigated the effect of EGCG on the constitutive expression of P65, pP65, IkBa, and pIkBa in KM3 cells. As shown by immunoblotting analysis, EGCG treatment resulted in a decrease in P65 and pP65 protein expression. P65 protein expression decreased to 0.9, 0.6, and 0.5 fold compared with the control group and pP65 protein expression decreased to 0.8, 0.5, and 0.4 fold compared with the control group in the dosedependence study, respectively [Fig. 2(A)]. Additionally, PARP cleavage analysis showed that the full-size PARP (116 kDa) protein was cleaved to yield an 85 kDa fragment after EGCG treatment. In the time-dependence study, the expression of P65 protein decreased to 0.9, 0.7, and 0.6 fold compared with the control group and

pP65 protein decreased to 0.6, 0.4, and 0.1 fold compared with the control group, respectively. PARP cleavage analysis also showed that the full-size PARP (116 kDa) protein was cleaved to yield an 85 kDa fragment after treatment of cells with EGCG [Fig. 2(B)]. As for IkBa and pIkBa, we found that the expression of protein IkBa increased whereas pIkBa decreased both in a dose- and time-dependent fashion. The expression of protein IkBa increased to 2.0, 2.8, and 3.7 folds compared with the control group (0 mM), respectively, in the dose–effect study and increased to 1.2, 1.4, and 1.8 folds compared with the control group (0 h), respectively, in the time–effect study [Fig. 2(C,D)]. On the contrary, the expression of protein pIkBa decreased to 0.8, 0.6, and 0.5 fold compared with the control group (0 mM), respectively, in the dose –effect study and Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 1021

Potentiation of EGCG-induced apoptosis by bortezomib in multiple myeloma cells

Figure 2 EGCG inhibited NF-kB/P65 expression, downregulated pIkBa expression, and upregulated IkBa expression in KM3 cells (A) Dose– effect of immunoblotting analysis of the protein P65, pP65, and PARP expression. (B) Time– effect of immunoblotting analysis of the expression of P65, pP65, and PARP. (C) Dose– effect of immunoblotting analysis of the expression of protein IkBa and pIkBa. (D) Time– effect of immunoblotting analysis of the expression of protein IkBa and pIkBa. The corresponding GAPDH levels are shown as loading controls. Data are expressed as mean + SD from three individual experiments. *P , 0.05 and **P , 0.01 compared with the control group.

decreased to 0.8, 0.7, and 0.5 fold compared with the control group (0 h), respectively, in the time–effect study [Fig. 2(C,D)]. Further, we studied the P65 gene expression pattern by real-time PCR. In the dose –effect study, the expression of P65 gene decreased to 0.10, 0.22, and 0.48 fold compared with the control group, respectively. There were significant differences between treatment Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 1022

groups and control group [Fig. 3(A)]. The expression of P65 gene decreased to 0.11, 0.22, and 0.58 fold at 12, 24, and 48 h compared with the control group, respectively [Fig. 3(B)]. These observations suggested that EGCG inhibits the expression of P65 gene and results in the degradation of protein P65 and pP65. At the same time, EGCG inhibits phosphorylation and degradation of IkBa.

Potentiation of EGCG-induced apoptosis by bortezomib in multiple myeloma cells

Figure 3 EGCG inhibited the expression of NF-kB/P65 mRNA (A) Dose– effect of EGCG-induced downregulation of P65 mRNA expression. Total mRNA was extracted from cells treated as in Fig. 1(A) and the expression level of the P65 was evaluated by SYBR Green real-time PCR. Amplification of 18S rRNA was performed as an internal standard. Data are expressed as mean + SD from three individual experiments. **P , 0.01 compared with the control group. (B) Time– effect of EGCG-induced downregulation of P65 gene at mRNA level. Total mRNA was extracted from cells treated as in Fig. 1(B) and the expression level of the P65 gene was evaluated using SYBR Green real-time PCR. Amplification of 18S rRNA was performed as an internal standard. Data are expressed as mean + SD from three individual experiments. **P , 0.01 compared with the control group.

EGCG promotes KM3 cell apoptosis through activation of caspase-3, -8, and -9 We used antibodies that specifically recognize the proand active forms of caspases (for caspase-3 and -8). EGCG treatment resulted in a dose-dependent increase in the cleaved product of casapse-3 (17 kDa), with concomitant decrease in the pro-form (32 kDa). Similarly, a dose-dependent increase in the active forms of caspase-8 (43 and 18 kDa) and of caspase-9 (35 kDa) was observed after EGCG treatment [Fig. 4(A)]. Further,

EGCG treatment also resulted in a significant timedependent increase in the active products of caspase-3, -8 and -9, respectively [Fig. 4(B)].

EGCG and bortezomib synergistically inhibit KM3 cell growth and induce apoptosis In the present study, we found that there are significantly synergistic effects on cell growth inhibition, induction of apoptosis, and activation of caspase-3, -8, -9, and PARP between the EGCG-treated group and the group treated

Figure 4 Immunoblotting analysis of caspase-3, -8, and cleaved caspase-9 expression (A) Dose– effect immunoblotting analysis of caspase-3, -8, and cleaved caspase-9 expression. (B) Time– effect immunoblotting analysis of caspase-3, -8, and cleaved caspase-9 expression. The corresponding GAPDH levels are shown as loading controls. Data are expressed as mean + SD from three individual experiments.*P , 0.05 and **P , 0.01 compared with the control group. Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 1023

Potentiation of EGCG-induced apoptosis by bortezomib in multiple myeloma cells

with EGCG and 20 nM bortezomib. There was a significant difference between the EGCG groups (0, 25, and 50 mM) and the groups in combination with bortezomib (P , 0.01, P , 0.01, and P , 0.01, respectively) [Fig. 5(A)]. Median dose–effect analysis of cell growth inhibition by EGCG with bortezomib yielded combination index values considerably ,1.0, indicating a highly synergistic interaction. The combination indexes for 25 and 50 mM EGCG with bortezomib were 0.58 + 0.13 and 0.52 + 0.12, respectively. The percentages of apoptosis in cells treated with EGCG (0, 25, and 50 mM), were 8.5%, 12.1%, and 29.5%, but increased to 9.0%, 25%, and 45.5% when combined with bortezomib (P , 0.05, P , 0.01, and P , 0.01, respectively) [Fig. 5(B)]. The combination indexes for 25 and 50 mM EGCG with bortezomib were 0.81 + 0.11 and 0.72 + 0.11, respectively. There was also a significant difference between the EGCG groups (0, 25, and 50 mM) and these groups combined with 20 nM bortezomib (P , 0.01, P , 0.01, and P , 0.01, respectively) in the decreased fold change in the expression of the P65 gene [Fig. 5(C)]. The combination indexes for 25 and 50 mM EGCG with bortezomib were 0.37 + 0.10 and 0.24 + 0.10, respectively. A similar synergistic effect occurred in the change of the proteins of P65, pP65, PARP, IkBa, pIkBa, caspase-3, -8, and cleaved caspase-9 (Fig. 6).

Discussion In recent years, EGCG has gained much attention due to its cancer chemopreventive properties. The tumorinhibiting property is well documented. Several studies have shown that EGCG treatment resulted in inhibition of cancer cell growth and induced apoptosis [10,12,15,25–27]. NF-kB consists of multiple members of the Rel family of proteins, which includes NF-kB ( p105/p50), NF-kB ( p100/p52), RelA ( p65), RelB, and c-Rel [28]. The NF-kB dimers are present in the cytoplasm in an inactive form bound to inhibitory subunits; upon activation, IkB is phosphorylated, which marks the inhibitor for ubiquitination and degradation by a proteasome-dependent pathway [29]. This process allows translocation of active NF-kB complexes into the nucleus, where they bind to specific DNA motifs in the promoter/enhancer regions of target genes and activate their transcription. Modulation in the transactivation domains and/or nuclear translocation of NF-kB by a variety of stimuli leads to cell cycle arrest and apoptosis [28]. Shammas et al. [30] found that EGCG induced both dose- and time-dependent growth arrest and subsequent apoptotic cell death in MM cell lines (INA6 and ARP cells), indicating that the 67-kDa laminin receptor 1 plays an important role in mediating EGCG activity in

Figure 5 EGCG combined with bortezomib (20 nM) inhibited KM3 cells growth and induced apoptosis (A) Cells were incubated with EGCG (25 or 50 mM) alone or combination with bortezomib (20 nM) for 48 h. All data are expressed as mean + SD from three individual experiments. **P , 0.01 compared with the control group. (B) Cells were treated as described in Fig. 3(A). Apoptosis was evaluated by Annexin V staining and FACScan analysis. The LR quadrant indicates the percentage of early apoptotic cells. (C) EGCG combined with bortezomib (20 nM) downregulated P65 mRNA expression. Total mRNA was extracted from cells treated as in Fig. 3(A) and the expression level of the P65 gene was evaluated by real-time PCR. Amplification of 18S rRNA was performed as an internal standard. Data are expressed as mean + SD from three individual experiments. **P , 0.01 compared with the control group. Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 1024

Potentiation of EGCG-induced apoptosis by bortezomib in multiple myeloma cells

Figure 6 Immunoblotting analysis of P65, pP65, PARP, IkBa, pIkBa, caspase-3, -8, and cleaved caspase-9 expression in KM3 cells treated with EGCG alone or in combination with 20 nM bortezomib (A) Immunoblotting analysis of P65, pP65, and PARP expression in KM3 cells treated with EGCG alone or in combination with 20 nM bortezomib. (B) Immunoblotting analysis of IkBa and pIkBa expression. (C) Immunoblotting analysis of caspase-3, -8, and cleaved caspase-9 expression. The corresponding GAPDH levels are shown as loading controls. Data are expressed as mean + SD from three individual experiments. *P , 0.05 and **P , 0.01 compared with the control groups (EGCG alone treatment).

MM. Evaluation of changes in the gene expression profile indicates that EGCG treatment activates distinct pathways of growth arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptordependent apoptosis (Fas ligand, Fas, and caspase 4), p53-like proteins ( p73 and p63), positive regulators of apoptosis, NF-kB activation (CARD10 and CARD14), and cyclin-dependent kinase inhibitors ( p16 and p18). In the present study, we have demonstrated that EGCG inhibits the expression of the P65 gene to result in the degradation of the protein P65 and pP65, meanwhile EGCG can inhibits phosphorylation and degradation of IkBa. EGCG also can actives caspase-3, -8, -9, and PARP, which leads to subsequent apoptosis of human myeloma KM3 cells. The inhibition of NF-kB transcriptional activity observed during EGCG-mediated apoptosis could be the result of two distinct inhibitory mechanisms: (i) inhibition of P65 and downregulation of pIkBa and upregulation of IkBa, which may disable the transactivation capacity; and (ii) activation of caspase-8, -9, -3, and PARP. There were time- and dose-dependent effects of EGCG on cell growth inhibition and induction of apoptosis. When EGCG was combined with bortezomib (20 nM), there was a synergistic effect. Bortezomib, a proteasome inhibitor, inhibits transcription factor NF-kB activation by protecting its inhibitor, IkBa, from degradation by the 26S proteasome. Degradation of IkB by proteasome activates NF-kB, which upregulates transcription of proteins that promote cell survival and

growth, decreases apoptosis susceptibility, influences the expression of adhesion molecules, and induces drug resistance in myeloma cells, is a promising novel agent for treatment of advanced MM. However, only 35% of patients with refractory disease in a Phase II study respond to bortezomib [31]. From the present study, EGCG can inhibit NF-kB/P65 and phosphorylation and degradation of IkBa and there is a synergistic effect when EGCG and bortezomib are combined. The results are different from Golden et al. [32]. They use a relative lower concentrations of EGCG (10 mM) and bortezomib (10 nM), whereas we used a relative high concentration of EGCG (25, 50, and 100 mM) and bortezomib (20 nM). We focused on the mechanism of EGCG inhibiting myeloma cell growth and inducing cell apoptosis potentiated by bortezomib, whereas they focused on the effect of EGCG on bortezomib in myeloma cells. So, we found that EGCG inhibits myeloma cell growth and induces cell apoptosis potentiated by bortezomib. This may be because of different drug concentrations or different cell lines. A relative study showed that 10 mM EGCG promotes RPMI8226 myeloma cell line growth but 20 mM inhibits its growth within 72 h but different results were found in MM1S, INA6, and OPM1 cell lines [29]. Given these previous findings, together with the findings of the present study, EGCG could be proven to be useful in the treatment of some types of cancer with EGCG alone or in combination with bortezomib, which represents a novel approach for MM therapy. Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 1025

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Funding This work was supported by a grant from the Natural Science Foundation of Guangdong Province, China (No. 2008-8151008901000064).

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