点击显示 收起
【摘要】 肿瘤多药耐药(multidrug resistance，MDR)是临床化疗成功最为严重的障碍。本文首先阐明了新拓扑异构酶II抑制剂沙尔威辛对MDR肿瘤细胞直接的细胞毒性作用及下调mdr-1基因和P-糖蛋白的作用。沙尔威辛能有效杀伤MDR细胞株，如K562／A02，KB／VCR和MCF-7／ADR细胞，其杀伤能力与对相应亲本细胞相当，而明显强于几种临床常用的抗癌药物。沙尔威辛下调mdr-1基因和P-糖蛋白的表达，但并不影响MRP和LRP基因。其次，揭示了转录因子c-jun的激活，在沙尔威辛下调K562／A02细胞内mdr-1基因表达及诱导凋亡过程中起着关键作用。沙尔威辛增加K562／A02细胞的c-jun表达明显早于其减少mdr-1基因的表达；c-jun反义寡核苷酸消除沙尔威辛升高c-jun蛋白、下调mdr-1基因表达的作用。沙尔威辛还促进JNK和c-jun磷酸化并增强转录因子AP1的DNA结合活性。此外，c-jun反义寡核苷酸还抑制沙尔威辛的凋亡诱导和细胞毒性作用。最后，进一步研究发现沙尔威辛本身不引起MDR表型。成功建立了对沙尔威辛具有8．91倍耐药的A549／SAL细胞株。该细胞株对抗代谢药产生6．70倍的耐药，但对多种其他天然来源的抗肿瘤药物、烷化剂以及铂类化合物则缺乏交叉耐药性。
【关键词】 沙尔威辛；多药耐药；mdr-1基因；转录因子c-jun；A549／SAL细胞
Exploration on anti-multidrug-resistant molecular mechanisms of salvicine and characterization of Salvicine.Salvicine -Resistant A549／SAL Cell Line
MIAO Ze-Hong， DING Jian.
Division of Anti-Tumor Pharmacology，State Key Laboratory of Drug Research，Shanghai Institute of Materia Medica，Chinese Academy of Sciences，Shanghai 201203，China
【Abstract】 Muhidrug resistance (MDR) is a major clinical problem in treating human cancers with conventional chemotherapeutic drugs．This study demonstrated that salvicine，a novel antitumor compound under clinical trial，exerted direct cytotoxicity against MDR tumor cells and down-regulated mdr-1／P-glycoprotein(P-gp) expression simultaneously．Salvicine effectively killed MDR sublines, such as K562／A02，KB／VCR and MCF-7／ADR，and parental K562，KB，and MCF-7 cell lines to an equivalent degree．Its cytotoxic activities were much more potent than those of several classical anticancer drugs．Salvicine induced the down-regulation of mdr-1 gene and P-gp expression，while not affecting MRP and LRP expression anti-MDR mechanism exploration revealed that transcription factor c-jun played a principal role in down-regulation of mdr-1 expression and induction of apoptosis by salvicine．Levels of c-jun expression were enhanced by salvicine prior to reduction of mdr-1 expression in K562／ A02 cells．Moreover，c-jun antisense oligodeoxynucleotides (AODs) prevented salvicine stimulated enhancement of c-jun protein and reduction of mdr-1 gene expression，but did not afect the increase in c-jun mRNA levels．Salvicine promoted phosphorylation of JNK kinase and c-jun protein and enhanced DNA binding activity of transcription factor AP1．Additionally， c-jun AODs also inhibited salvicine-induced apoptosis and cytotoxicity．Finally，salvicine was further shown not to induce a tumor MDR phenotype．We established a salvicine-resistant tumor cell subline，A549／SAL，which displayed 8．91-fold resistance to salvicine and an average of 6.70-fold resistance to the antimetabolites．The subline，however，was not resistant to alkylating agents，platinum compounds，and other naturally derived antineoplastics．
【Key words】 salvicine；multidrug resistance；mdr-1；c-jun；A549／SAL cells
Malignant tumors can become resistant to conventional antineoplastic agents, which has been a major clinical problem in treating human cancers．Tumor drug resis tance may be divided into two kinds，individual drug resistance and muhidrug resistance (MDR)．The complex mechanism of MDR is a sevel challenge for both basic and clinical researches on tumor therapy.
Salvicine is a novel diterpenoid quinone compound synthesized by the structural modification of a natural product lead isolated from a Chinese medicinal plant，Salvia priontis lance(Labiatae)［1］．It possesses potent anticancer activities in vitro and in vivo against various hematological and solid tumor cell lines［2］．and enters into clinical trial in China.Mechanism studies revealed that topoisomerase 11(TOPOⅡ)is the primary cellular target of salvicine［3］. The inhibitory activity of this compound as a TOPO II poison is derived from its ability to stabilize DNA strand breaks，either through interactions with the enzyme alone or the DNA—enzyme complex［4］．In the current study，we further demonstrated (1) salvicine—exerted direct cytotoxicity against MDR tumor cells and mdr-1／P-gp expression down—regulation；(2) the principal role of transcription factor c-jun in salvicine-induced mdr-I／P-glycoprotein (P-gp)expression inhibition and apopotosis，and (3) the non-MDR phenotype of tumor resistance to salvicine．
1 Salivicine-exerted direct cytotoxicity against MDR tumor cells and mdr·I／P．gp expression down-regulation［5］
1.1 In vitro cytotoxicity of salvicine in MDR tumor celIs Salvicine displayed significanl cytotoxicity in the three MDR sublines examined， with an average IC50 va1ue of 2．48μmol ·L-1, which was nearly equivalent to that observed with the corresponding parental cell lines (average IC50：1．91μmol ·L-1)．In contrast，the average IC50 values of reference drugs，doxoruhicin (DOX)，vincfistine(VCR)，and etoposide(VP16)were 5.56μmol ·L-1, 30.22μmol ·L-1 and 204.81μmol ·L-1，individually in three MDR sublines，compared to 0.036μmol ·L-1，0.47μmol ·L-1 and 2.28μmol ·L-1 in the parental partners．The cytotoxicity of salvicine against MDR tumor cells was much more potent than that of the three conventional anticancer drugs， DOX，VCR and VPI6，with an average resistantce factor of 1.42 for salvicine versus 233.19，344．35， and 71．22 for DOX, VCR, and VP16, respectively(Fig．1)．
Fig.1 Direct cytotoxicity of salvicine against MDR tumor cells（略）
1.2 Apoptosis induction in MDR K562／AO2 cells by salvicine Salvicine induced apoptosis in MDR K562／A02 and tile parental cell line in a similar leve1．Treatment with salvicine for 24h induced apoptesis to the same degree in both cell lines in a dose-dependent manner by flow cytometr. At salvicine concentrations lower than or equal to 4μmol ·L-1, apoptosis was not significant．Higher concentrations of salvicine resulted in significant apoptosis with an increase in percentage to 69.4％ and 59.1％ in MDR cells, and to 40.5％ and 75.6％ in parental ones at 8μmol ·L-1and 10μmol ·L-1，respectively．But the reference drug，DOX(up to 4μmol ·L-1) did not induce apoptesis in MDR cells，although significant apoptosis was observed in the parental cells at the same drug concentration．This result was confirmed by DNA agarose gel electrophoresis．Salvicine of 20μmol ·L-1and 40μmol ·L-1 induced an equivalent extent of DNA fragmentation in MDR K562／A02 and parental K562 cells．Additionally，salvicine-treated K562／A02 cells also displayed the dose-dependent morphological changes typical of apoptosis including apoptotic bodies, nuclear condensation，and cell shrinkage．
1.3 Salvicine down-regulating mdr-1 gene expression in MDR K562／A02 celIs MDR K562／A02 cells were shown by RT-PCR to express mdr-1 and MRP genes, but lack LRP mRNA, while none of the three genes were detectable in parental cells．Treatment with salvicine(l～l6μmol·L-1) for 24h led to a dose-dependent down-regulation of mdr- 1mRNA expression in MDR cells．Levels of mdr-1mRNA nearly completely disappeared in cells treated with 16μmol ·L-1 salvicine. No evident change in MRP mRNA expression was observed in salvicine-treated K562／A02 cells up to concentrations of 8μmol ·L-1. Consistetly，the P-gp levels progressively decreased in the MDR cells treated with increasing concentrations of salvicine for 24h(Fig．2)．
Fig.2 Salvicine reduced mdr-1/P-gp expression in MDR K562/A02 clls（略）
1.4 Salvicine activating caspase-1 and caspase-3, but not caspase-8 in MDR K562／A02 cells Salvicine activated caspase-1and caspase-3, but not caspase-8, in both cell in a time and dose-dependent manner．Treatment with 20μmol ·L-1 salvicine for 12 h lead to an increase in the activities of caspase-1 and caspase-3 of MDR K562／A02 and parental cells, with another sharp increase upon exposure for 24h．Moreover，pretreatment with 100μmol·L-1 caspase-1, caspase-3 and pancaspase inhibitors for 1h a1most completely blocked salvicine-induced apoptosis in both MDR K562／A02 and parental cells．The resuIts confirmed caspase-1 and caspase-3 activation by salvicine, and further provided evidence of caspase dependency in salvicine induction of apotosis in MDR and parental K562 cells．
1.5 Salvicine enhancing the ratio of bax to bcl-2 mRNA by down-regulating bd-2 mRNA expression in MDR K562／AO2 cells Salvicine reduced bcl-2 mRNA expression、whereas no obvious changes were noted in bax mRNA levels in MDR K562／A02 cells treated with 4μmol ·L-1 and 8μmol ·L-1 salvicine for 24 h．The ratio of bax to bel-2 mRNA was enhanced from l.03 at 2μmol ·L-1 to 1.39 at 8μmol ·L-1．The results suggest that salvicine possibly triggers caspase-1 and caspase-3(at least partly)by inhibiting bcl-2 gene expression．
2 The principal role of transcription factor c-jun in salvicine-induced mdr-1／P-glycoprotein expression inhibition and apoptosis［6］
2.1 Enhancement of c-jun mRNA levels prior to reduction of mdr-1 mRNA levels Following treatment of MDR K562／A02 and parental K562 cells with 20μmol·L-1 salvicine，c-jun mRNA levels first rose then decreased in both cell lines．In MDR cells, c-jun mRNA levels rose up to 1.24 times the basal value at 0.5h, reached their maximum of about 1.44-times basal at 2 h，and remained high before beginning to decrease at 12h, and returned to near control levels at 16 h．A similar phenomenon was observed in parental K562 cells, where c-jun mRNA levels reached a maximum of 1.73-times basal at 12 h．On the other hand，levels of mdr-1 mRNA changed little before 8 h，declined after 12 h，and went down to 36.7％ of basal in K562／A02 cells at 16 h．In parental K562 cells, no mdr-1 mRNA was detectable．
The same trend was observed when cells were exposed to gradient concentrations of salvicine for fixed durations．In the range of 5μmol ·L-1 to 40μmol ·L-1，salvicine elevated c-jun mRNA levels in K562／A02 and K562 cells．but did,not significantly affect mdr-1 mRNA at 2 h．At 16 h,5μmol ·L-1 salvicine enhanced c-jun mRNA levels in MDR cells，and at higher concentrations，c-jun mRNA levels reduced gradually while mdr-1 mRNA levels decreased progressively with increasing concentrations． These results indicate that salvicine-stimulated decreases in mdr-1 mRNA levels occur after increases in c-jun mRNA levels in MDR cells．
2.2 Salvicine raising c-jun，and lowering P-gp protein expression Following treatment of both MDR and parental cells with 20μmol·L-1 salvicine，c-Jun protein levels began to rise as early as 0.5 h．In contrast to the changes at the mRNA level，c-Jun protein reached a plateau after 12 h and remained high to 24 h．This difference may be due to a shorter half-life of c-jun mRNA compared to protein．A decline in P-gp protein levels was observed，consistent with that of mdr-1 mRNA levels in salvicine-treated K562／A02 cells．Levels of c-jun increased concentration-dependendy in both cell lines treated with salvicine for 24 h，but levels of P-gp decreased progressively at concentrations higher than 10μmol·L-1 in MDR cells．As was the case for mRNA levels，enhancement of c-jun protein levels occurred earlier than the decline in P-gp protein levels in MDR cells．
2.3 Increased phosphorylation of JNK kinase and c-jun protein We investigated whether salvicine-induced c-jun protein was in its active，phosphorylated form (p-c-jun)，and whether this process involved phosphorylated JNK(p-JNK)．Treatment of either MDR or parental cells with salvicine lead to increased levels of P-JNK and P-c-jun, the amounts of which reflected incubation time and salvicine concentration．Basically，these changes paralleled those in the levels of c-jun．In contrast，the total amount of JNK did not change．The results indicate that salvicine stimulated phosphorylation of c-jun at serines 63 and 73 by activating JNK kinase in a time-and concentration-dependent manner．
2.4 c-jun AODs rescuing reduction of mdr-1 gene expression following activation of c-jun Pretreatment of cells with 50μg／ml c-jun antisense oligodeoxynucleotides (AODs) for 1 h did not change the response pattern of c-jun mRNA to salvicine，but altered the levels of c-jun protein，mdr-1 mRNA and P—gp protein．In both c-jun AODs-treated and-untreated groups, c-jun mRNA levels still rose at 2 h and declined approximately to control values at 16 h in salvicine-treated K562／A02 and K562 cells． Contrarily，c-jun AODs completely reversed the reduction of mdr-1 mRNA by salvicine in MDR cells．At the protein level，c-jun AODs prevented upregulation of c-jun, and p-c-jun，and the down regulation of P-gp by salvicine. These data further demonstrate that enhancement of c-jun expression might lead to down regulation of mdr-l expression in salvicine-treated MDR cells(Fig．3)．
Following pretreatment with 50μg/ml c-jun AODs or c-jun SODs for 1h,both MDR and
parental K562 cells (5×105/ml) were exposed to 20μmol·L-1 salvicine for (A) 2h or 16h,or (B) 24h
Fig.3 Effect of c-jun AODs on expression of c-jun and mdr-1 genes in MDR K562/AO2 cells（略）
2.5 Salvicine elevating the DNA binding activity of AP1 We further studied the eftect of salvicine on API DNA binding activity using ELISA(enzyme-linked immunosorbent assay)．The nuclear extract derived from pborbol 12-myristate l3-acetate(TPA)-treated human T lymphocyte leukemia Jurkat cells was used as a positive contro1．We observed that nuclear extracts from K562／A02 cells treated with 20μmol·L-1and 40μmol·L-1 salvicine for 24 h contained a binding activity to the AP1 DNA binding consensus sequence，whereas those from untreated K562／A02 cells did not．The results were consistent with increased levels of c-jin and p-c-jun and decreased levels of mdr-l mRNA and P-gp protein．Interestingly，it appears the components of APl in salvicine-treated K562／A02 cells might be different from those in TPA-treated Jurkat cells because band shift 1 lagged significantly behind band shift 2．
2.6 c-jun AODs inhibiting salvicine-induced apoptosis and cytotoxicity After l h preincubation with 50μg/ml c-jun SODs or A0Ds, cells were treated with 20μmol·L-1 salvieine for 24 I1．DNA agarose gel electophoresis showed that salvicine-induced apoptosis was clearly inhibited by c-jun AODs，but not by SODs．Consistent with this，pretreatment with c-jun AODs also dramatically decreased the salvicine cytotoxicity against both cell lines，as determined by the M’IT assay． Pretreatment with c-jun AODs caused the growth inhibitory rate elicited by salvicine to decrease from 64.64％ to 9.53％ in MDR K562／A02 cells, and from 67.48％ to 23.82％ in parental cells．It is worth noting that the degree of growth inhibition of resistant cells was significantly greater than that of parental cells. These results demonstrate that alteration of c-jun expression is a critical feature of salvicine-stimulated apoptosis and cytotoxicity(Fig．4)．
Fig.4 Pathways involved in the activation of c-jun by salvicine（略）
3 The non-MDR phenotype of tumor resistance to salvicine［7］
3.1 Establishment of A549／SAL and its resistance to salvicine We have previously shown that A549 cells respond well to salvicine，both in vitro and in vivo． In the present study, we established a salvicine-resistant subline，denoted A549／SAL， with a resistance factor of 8.91，by 45 passages of A549 cells through stepwise increases in salvicine concentration，from 15μmol·L-1to 35μmol·L-1, plus a final single-step selection with 80μmol ·L-1 salvicine．When incubated continuously in salvicine-free medium for more than 1 month，this A549／SAL cell line maintained its resistance to salvicine，indicating its stability．
3.2 Cross-resistance profile of A549／SAL to representative antineoplastic drugs A549／SAL cells were cross-resistant to the antimetabolites, including fluorouracil，gemcitabine，and methotrexate, with an average resistance factor of 6.70．These cells，however，lacked resistance to the naturally-derived anticancer drugs．including the Topo II inhibitors doxorubicin and etoposide，the Topo I inhibitor hydroxycamptothecin，and the antimicrotubule drugs taxol and vincristine，all of which are subtrates of P—gp．In addition, these A549／SAL cells remained nearly as sensitive to the alkylating agents chlormethine and melphalan and to the platinum compound cisplatin as their parental A549 cells．The resistance profile of this sub-line suggests that it does not possess an MDR phenotype(Fig．5)．
3.3 Induction of apoptosis by salvicine in A549／SAL cells While exposure of both A549 and A549／SAL cells to salvicine for 24h lead to concentration—dependent apoptosis，the degree of apoptosis induced by a given concentration of salvicine was lower in the A549／SAL than in the parental cells．Especially at 40μmol·L-1salvicine, the yellowish green stains were much fewer and smaller in the A549／SAL group than in the A549 group．We also observed that salvicine of 10μmol·L-1, which lead to growth inhibition of about 80％ in A549 cells and was used to maintain the drug resistance of A549／SAL cells, resulted in A549 cell apoptosis in a time-dependent manner, in contrast to the insignificant changes in the salvicine-resistant cells．
3.4 Biological properties of A549／SAL The selective pressure induced by long-term exposure to salvicine resulted in great changes in A549 morphology and growth rate．In sharp contrast to the colony-growing tendency of the irregular polygonal parental cell, A549／SAL cells tended to grow primarily in a scattered manner and possessed oval or fibroblast-like shapes．A549／SAL cell also grew more slowly than the parental cells，with a doubling time of(28.74 ±2.23)h，compared with a doubling time of(23.41±0.68)h for the A549 cells．The results suggested that，compared with parental cells，the A549／SAL cell line had a longer cell cycle and increased mobility．
Thus，we further assayed the ability of A549／SAL cells to migrate and invade in vitro using the Boyden chamber assay．Much more A549／SAL cells than A549 cells passed through the upper membrane of the transwell inserts．regardless of whether the inserts were pretreated with MatrigelTM.These findings were confirmed by spectrophotometric analysis，which gave OD values 1.68 times higher for A549／SAL migration and 1.78 times higher for A549／SAL invasion，when compared with parental cells．
Fig.5 Drug resistance profile of A549/SAL cells（略）
3.5 Expression of drug-resistance-related RT-PCR analysis showed that transcripts of the drug-transporting-protein gene MRP, the drug action target genes Topo II α and β，the proliferation related gene PCNA，and the drug-metabolism enzyme gene GST, were expressed equally in A549／SAL and A549 cells．Long—term exposure of A549 cells to salvicine also did not alter the expression of p21，gadd45，bcl-2，and bcl—xL mRNAs．In contrast，the level of expression of p53 and bax mRNAs was lower，and the level of expression of mdm2 mRNA was higher，in A549／SAL than in A549 cells．We did not detect mRNAs encoded by the mdr-1 and bcl-2 genes in either cell line．This mRNA expression pattern indicates that salvicine，a naturally-originated Topo II inhibitor，did not induce expression of MDR related genes，nor did it suppress the expression of drug action-target genes．
We also assayed the expression of p53 and p53-related proteins in A549／SAL by Western blot analyses．Consistent with our RT-PCR findings，the level of p53 and bax protein expression was much lower，and the level of expression of MDM2 protein was much higher，in A549／SAL cells compared to A549 cells.Western blot analysis also showed that the levels of p21 and GADD45 proteins were approximately the same in the two cell lines．These findings suggest that resistance to salvicine may be related to a disruption in the down stream apoptotic signal pathway involving p53 and p53-related genes．
Our study reaches the following conclusions：firstly，salvicine exerts the potent anti-MDR activity against MDR tumor cells by inhibiting of mdr-1／P-gp expression；secondly，transcription factor c-jun plays a principal role in down regulation of mdr-1 expression and induction of apoptosis in salvicine treated human MDR K562／A02 cells；and finally，vitro tumor resistance to salvicine belongs to individual，not multiple，drug resistance．These data lay a solid biochemical basis for possible clinical application of salvicine alone, or in combination with conventional antineoplastics in treating MDR tumors and also provide new insights into the complicated regulatory mechanism of mdr-1 gene expression．
【References】
1 Zhang JS，Ding J，Tang QM，et al.Synthesis and antitumor activity of novel diterpenequinone salvicine and the analogs．Bioorg．Med．Chem．Lett,1999,9：2731-2736.
2 Qing C，Zhang JS，Ding J．In vitro cytotoxicity of salvicine，a novel diterpenoid quinone．Acta Pharmacol Sin，1999，20：297-302.
3 Meng LH，He XH，Zhang JS，et al.DNA topoisomerase II as primary cellular target for salvicine in Saccharomyces cerevisiae．Acta Pharmacol Sin,2001,21：741-746.
4 Meng LH，Zhang JS，Ding J．Salvicine，a novel DNA topoisomerase II inhibitor，exerting its efect by trapping the enzyme-DNA cleavage complex．Biochem．Pharmaco1,2001,62：733-74l.
5 Miao ZH，Tang T，Zhang YX，et al.Cytotoxicity，apoptosis induction and down regulation of mdr-1 expression by the anti-topoisomerase II agent．salvicine，in multidug-resistant cells．Int J Cancer，2003，106：l08-115.
6 Miao ZH，Ding J．Transcription factor c-jun activation represses mdr-1 gene expression．Cancer Res，2003，63：4527-4532.
7 Miao ZH，Tong LJ，Zhang JS，et al.Characterization of salvicine-resistant lung adenocarcinoma A549／SAL cell line．Int J Cancer,2004,110：627-632.
(编辑：余 强)
作者单位： 201203 上海，中国科学院上海药物研究所新药研究国家重点实验室肿瘤药理组