Biomedicine & Pharmacotherapy 66 (2012) 368–372

Original article

Genetic protein TmSm(T34A) enhances sensitivity of chemotherapy to breastcancer cell lines as a synergistic drug to doxorubicin

Yuxin Xu a,1, Wenyun Zheng b,1, Tianwen Wang c, Ping Wang a, Ling Zhu a, Xingyuan Ma a,*a School of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, Chinab School of Pharmacy, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, Chinac School of Biotechnology, Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, Jiangsu, China

A R T I C L E I N F O

Article history:

Received 4 December 2011

Accepted 15 December 2011

Keywords:

Genetic protein TmSm(T34A)

Enhancing sensitivity

Chemotherapeutic agent

Breast cell lines

A B S T R A C T

In order to eliminate common side effects to cancer patients and resistance from chemotherapy, a genetic

protein TmSm(T34A) was investigated as a sensitizer to doxorubicin. The results indicated TmSm(T34A)

enhanced the sensitivity of three breast cancer cell lines to doxorubicin with low dose, and reduced the

dose of doxorubicin significantly in contrast to common effective dose. As a synergistic therapy, the

TmSm(T34A) also caused strongest apoptotic activity in MCF-7, and the possible molecular mechanisms

were explored primarily. The research showed the TmSm(T34A) is promising to be a potential drug in

strengthening therapy effects of breast cancer chemotherapy.

� 2012 Elsevier Masson SAS. All rights reserved.

Available online at

www.sciencedirect.com

1. Introduction

The most frequent cancer of women in developed countries isbreast cancer. Although many patients have been cured bychemotherapy, the side effects from chemotherapy to cancerpatients cannot be ignored. For example, it may induce alopecia,liver and kidney damages, premature ovarian failure and othervarious adverse reactions to multiple systems of whole body [1].More importantly, the resistances from chemotherapeutic agentsare arousing more and more concerns. It was reported that morethan 90% patients with metastatic cancer suffered failure oftreatment because of drug resistance [2]. Therefore, overcomingthe resistance of drug was crucial to effective treatment of breastcancer, and a new strategy involved cutting down the dosage of thechemotherapeutic drug and enhancing cell sensitivity would begood optimization program to solve these problems.

Survivin is high-expressed especially in malignant cancer andtissues, such as breast, lung, colon, tummy, oesophagus, pancreas,liver, bladder, uterine, ovary and so on [3–6], but is rarelyexpressed in normal differentiated tissues. Survivin is a nodalprotein because of its involvement in multiple pathways of cellularhomeostasis. It has been reported that it promotes cell prolifera-tion, confers resistance cell death, regulates cell division,modulates apoptotic and non-apoptotic cell death, regulates cellsurvival in the face of unfavorable milieus and acts as a resistance

* Corresponding author. Tel./fax: +86 2164252257.

E-mail address: xymy@ecust.edu.cn (X. Ma).1 Contributed equally to this work.

0753-3322/$ – see front matter � 2012 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.biopha.2011.12.004

factor to various anticancer therapies [7–9]. So targeting the nodalproteins was a better choice than inhibiting single molecules, somestrategies to inhibit survivin have been reported, such as anti-sense oligonucleotides, ribozymes, small interfering RNAs, domi-nant-negative mutants, but all of these methods targeting survivinwere on gene level inhibition [10,11].

The recombinant protein TmSm(T34A), abbreviation of TATm-Survivin(T34A), has been designed and prepared by geneticengineering in previous experiment and later optimization inour laboratory [12,13]. A series of results revealed thatTmSm(T34A) could induce apoptosis to various cancer cell linesin early stage and suppressed proliferation. It was reported thatsurvivin phosphorylation was essential for its protective function,stimulating spontaneous mitochondrial apoptosis and increasingchemo-susceptibility to taxol [14]. This aroused us to explorethe possibility and application of the recombinant proteinTmSm(T34A) to enhance sensitivity of chemotherapy and eluci-date its related molecular mechanisms in the research further.

2. Materials and methods

2.1. Cell lines and cell culture

Human breast cell lines MCF-7, T-47D and Bcap-37 werecultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco,USA) supplemented with 10%(v/v) fetal bovine serum (Gibco, USA),100 mg/ml streptomycin and 100 unit/ml penicillin. The mediumof MCF-7 and T-47D were added insulin as subcultures. Cells wereincubated at 37 8C with 5% CO2 supply.

Fig. 1. The effect of doxorubicin on proliferation of breast cancer cell lines: a: Bcap-

37; b: T-47D; c: MCF-7 cells. These three cell lines were treated for 48 h with

1.117 ug/ml concentrations of TmSm(T34A). MTT assay was used to determine

growth relative to control cells. Data are expressed as percentage of control and

mean � SD of each treated cell lines.

Y. Xu et al. / Biomedicine & Pharmacotherapy 66 (2012) 368–372 369

2.2. Cell viability assay

MCF-7, T47-D and Bcap-37 cells in 96-well plate (1-5 � 104 cells/well) were divided into three groups, blank control group,doxorubicin-treated groups and doxorubicin–TmSm(T34A)-treatedgroup. The treatment group cell were incubated with differentconcentration of doxorubicin or doxorubicin–TmSm(T34A), respec-tively. The control cells were incubated with phosphate buffersolution (PBS buffer) instead of drug. After incubation, thesupernatant was cleared away, 180 mL medium and 20 mL MTTsolution (5 mg/ml) were added and incubated for 4 h, MTT-containing medium was removed and 150 mL DMSO was addedto each well to dissolve formazan. The optical densities of thesamples were determined by a spectrophotometer at 490 nm. Eachexperiment was performed independently for at least three times.

2.3. Cell cycle and apoptosis assay

MCF-7 cells (5 � 105 cells/well) were seeded into 6-well dishes(corning, Elmira, NY) overnight. After treated with doxorubicin atthe concentration of its IC50, TmSm(T34A), or doxorubicin–TmSm(T34A) for 48 h, the cells were harvested at 1000 rpm for10 min, and then washed with PBS twice. The cells for cell cycleassay were fixed by ice-cold 70% (v/v) ethanol in 1 � PBS (PH7.4) at4 8C overnight and incubated with RNase A (1 mg/ml) at 37 8C for30 min after centrifugated at 1000 rpm for 10 min, then werestained with propidium iodide (PI) (0.05 mg/ml) for 10 min. Theprepared samples were analyzed by flow cytometry. The cells forapoptosis assay were resuspended in 500 ml of 1X Binding Buffer;5 ml Annexin V-FITC and 5 ml PI (50 mg/ml) were added forstaining 5 min at room temperature in the dark, then flowcytometry analysis was carried out.

2.4. Western blot

Cells were plated onto 60-mm dishes at a density of 5–20 � 105/well. The adherent cells were treated by drug for planned time andwashed twice with PBS. The cells were harvested by centrifugationand washed twice with PBS, and then lysized in 200 mL RIPA buffer(50 mM Tris–HCl, pH7.4, 1%NP-40, 50 mM NaCl, 0.1%SDS, 1% Na-deoxycholic acid, 1 mM sodium or thovanadate, 2 mM PMSF) for30 min on ice with gentle rocking. The total protein concentrationwas determined by the BCA protein assay with BSA as standard. Fiftyto 70 mg cells lysate protein were separated by 12% SDS-PAGE andthen transferred onto PVDF membranes. The membranes wereblocked for 2 h at room temperature with 5% non-fat dry milk in PBScontaining 0.05% Tween-20 (PBST), and then probed with antibodiesagainst caspase-9 (Biovision, USA), PARP (Biovision, USA), Bcl-2(Bioworld, Cambridge, UK) or b-actin (Sigma-Aldrich) for 1 h atroom temperature. Then the membranes were washed with PBST forthree times, and incubated with the secondary anti-mouse rabbitIgG-HRP for 1 h at room temperature. Finally, blots were visualizedon X-ray film with enhanced ECL western blotting detection reagents.

2.5. Statistical analysis

The data were expressed as the mean � SD based on the resultsobtained from at least three independent experiments. A level of P

less than 0.05 was considered to be statistically significant.

3. Results

3.1. TmSm(T34A) increase the sensitivity of the cells to doxorubicin

In the present study, the anticancer effect of doxorubicin ordoxorubicin–TmSm(T34A) combination were determined by the

MTT assay on three human breast carcinoma cell lines: MCF-7, T-47D and Bcap-37. Doxorubicin exerted a dose-dependent toxicityagainst the three cell lines tested (Fig. 1). MCF-7 was most sensitiveto doxorubicin with an IC50 of 0.462 � 0.005099 mg/ml, the IC50 forT-47D and Bcap-37 was 0.507 � 0.036341 mg/ml, and 0.5644 �0.14659 mg/ml, respectively. 1.117 ug/ml is non-toxic dosage towardthe three cells above. Compared to the doxorubicin-treated group, thecombined administration of TmSm(T34A) and doxorubicin resulted ina decrease in IC50 about 47.2%, 36.7%, 15.8% for Bcap-37, T-47D andMCF-7, respectively, shown in Table 1. So TmSm(T34A) was thought toimprove the sensitivity of the cells to doxorubicin remarkably.

3.2. Effects of TmSm(T34A) on apoptosis and cell cycle of MCF-7

Annexin V-FITC/PI experiment was carried out to further testthe effect of sensitization and apoptosis from the TmSm(T34A). Asshown in Fig. 2, the apoptosis rate was 42.5% when treated alonewith doxorubicin, and that increased to 50.19% when treated with

Fig. 2. The synergistic effect of doxorubicin and doxorubicin combined with

different concentration (0.058 mg/ml, 1.117 mg/ml, 3.34 mg/ml) TmSm(T34A) for

24 h, 48 h, and 72 h in MCF-7 cells. The inhibition rate was measured by MTT assay.

Results were shown in the value of absorbance at 490 nm as mean � SD of each

treatment.

Table 1The IC50 changing of TmSm(T34A) increase anticancer sensitivity of doxorubicin by

MTT.

IC50 (ug/ml) Doxorubicin Combination

Bcap-37 0.5644 � 0.146 0.475 � 0.109

T-47D 0.507 � 0.036 0.3207 � 0.0712

MCF-7 0.462 � 0.005 0.244 � 0.011

Bcap-37, T-47D and MCF-7 treated by doxorubicin and doxorubicin combined with

TmSm(T34A), then used SPSS 11.5 to calculate IC50, represented as mean � SD.

Y. Xu et al. / Biomedicine & Pharmacotherapy 66 (2012) 368–372370

doxorubicin and TmSm(T34A) combination. And it was notewor-thy that, 1.117 ug/ml TmSm(T34A) alone had no effect onapoptosis in MCF-7 cells. Thus, it demonstrated that TmSm(T34A)might increase doxorubicin-induced apoptosis in MCF-7 cells andsubsequently lead to an increasing cytotoxicity of doxorubicin. Itproved TmSm(T34A) could sensitize the breast cancer cell lines todoxorubicin.

Fig. 3. TmSm(T34A) treatment increase doxorubicin-induced apoptosis in MCF-7 cells w

treated with TmSm(T34A) alone. A3, B3: cells were treated with doxorubicin alone. A4

above were treated for 48 h.

It was also found that doxorubicin arrested cell cycle atG2/M phase in MCF-7 cells, while combined treatment withTmSm(T34A) led only to small increase in cell population at theG2/M phase from 63.97 to 67.46%, as shown in Fig. 2. These resultssuggested that TmSm(T34A) treatment might not potentiate theability of doxorubicin through G2/M arresting to synergisticapoptosis.

3.3. The sensitization is dose-dependent and time-dependent

The most sensitive MCF-7 cell line was treated with thedoxorubicin and different concentrations of TmSm(T34A) combina-tion, combination A (0.056 ug/ml doxorubicin–0.058 mg/mlTmSm(T34A)), combination B (0.056 ug/ml doxorubicin–1.117 mg/ml TmSm(T34A)), combination C (0.056 ug/ml doxorubi-cin–3.34 mg/ml TmSm(T34A)), for 24 h, 48 h and 72 h respectively.The results showed the sensitization caused by TmSm(T34A) wastime-dependent and dose-dependent, shown in Fig. 3. After the cellswere incubated with doxorubicin alone for 72 h, the inhibition ratewas twice than that for 24 h, and in ADM + 1.117 ug/mlTmSm(T34A) group the 72 h was 2.6-fold compared with 24 hinhibition rate. When the cells were incubated with doxorubicin and3.34 ug/ml TmSm(T34A) for 72 h, the inhibition was three timesstronger comparing with cells treated by doxorubicin alone for 72 h.We next checked the dosage increase of TmSm(T34A) enhanceddoxorubicin sensitivity, ADM + 0.058 ug/ml TmSm(T34A) groupinhibition rate for 72 h was 2.7-fold compared with ADM groups, theinhibition of ADM + 3.34 ug/ml TmSm(T34A) for 72 h, was threetimes comparing with ADM groups for the same time. So we furtherconcluded, the sensitization of TmSm(T34A) is dose-dependent andtime-dependent.

3.4. The effect of doxorubicin and TmSm(T34A) on proteins related

apoptosis in MCF-7

To confirm that the main apoptotic pathway(s) involved thesynergistic cytotoxicity of doxorubicin administrated in combina-tion of TmSm(T34A) and doxorubicin on MCF-7 cells, the

ithout G2/M arresting. A1, B1: cells were treated without drug. A2, B2: cells were

, B4: both cells were treated with doxorubicin combination with TmSm(T34A). All

Fig. 4. Effect of TmSm(T34A) on the doxorubicin-treated MCF-7 cells. The level of

Caspase-9, PARP and Bcl-2 expression, followed treatment with TmSm(T34A) singly

or with doxorubicin singly, the synergistic effects of doxorubicin and TmSm(T34A)

for 48 h were determined in cells by western blotting. Actin was measured as the

quantity control.

Y. Xu et al. / Biomedicine & Pharmacotherapy 66 (2012) 368–372 371

expression of some apoptosis associate proteins, caspase-9, PARPand Bcl-2 were analyzed. The expression of caspase-9 and cleaved-caspase-9 were determined by western blot (Fig. 2). The treatedcells with TmSm(T34A) or doxorubicin up-regulated cleaved-caspase-9 activity to 1.5- and 4.7-fold, respectively, compared tothe control. The combination treatment boosted cleaved-caspase-9activity up to 5.3-fold. However, caspase-9 was descended in thethree treatment groups. The treatment with doxorubicin orTmSm(T34A) resulted in an increase of cleaved-PARP (Fig. 2),3.44-fold for doxorubicin, 1.86-fold TmSm(T34A), and 4.5-fold fordoxorubicin-TmSm(T34A) combination for 48 h. Meanwhile theexpression of PARP was improved by 0.82-, 1.31-, 1.54-fold. Tospecify additional molecular mechanisms of doxorubicin andTmSm(T34A) inducing apoptosis, the changes of Bcl-2 was alsoexamined, showing a synergistic decrease, shown in Fig. 2. Bcl-2showed a cute decrease in treated groups. Especially in doxorubi-cin combination treated group, Bcl-2 decreased sharply, shown inFig. 2. Bcl-2 decreasing may be the reason that reduces theresistance of tumor to anticancer drugs and increases thesensitivity. The change may be potentially reducing tumorresistance to anticancer drugs.

4. Discussion

4.1. Targeted inhibition to survivin is a good anticancer strategy in the

level of protein

The research revealed silencing survivin and increasing thesensitivity of cancer cells to chemotherapeutic agents coulddecrease drug dose requirement significantly. Therefore, thesurvivin-directed recombinant protein combined of chemothera-peutic agent was proved to be a valuable therapy strategy and aneffective approach for cancer treatment. The studies and relatedmechanisms on inhibiting survivin to enhance sensitivity ofchemotherapy had been reported repeatedly in level of gene inrecent years. For example, the plasmid vector containinghammerhead ribozymes targeting survivin mRNA were trans-fected into melanoma cells, resulting in decreased expression ofsurvivin protein and an increased caspase-9-dependent apoptoticresponse to cisplatin and topotecan [15,16]. Furthermore, thesurvivin antisence oligonucleotides enhanced anticancer activityagainst established therapies [17], and down-regulation ofsurvivin expression in cancer cells also could enhance sensitivityto other anticancer agents, such as TRAIL, cisplatin, taxol andetoposide [1,18–20]. In addition, it was reported that down-regulation of survivin expression by antisence oligonucleotidescould also increase sensitivity of cancer cells to radiation treatment[21]. All above evidences indicated that down-regulation ofsurvivin by (combined) therapy was a good choice for cancertreatment. However, the effect of exogenous recombinant proteinon enhancing sensitivity to chemotherapy was reported andconfirmed firstly in our work, which may be more significant andhelpful to develop a supplementary or synergistic therapy drug ofdoxorubicin or other chemotherapeutic agents, even and radiationtreatment for cancer.

4.2. Recombinant protein TmSm(T34A) has multifunctions to breast

cancer cells

Survivin is overexpressed in numerous tumor types includinghuman bladder cancer and breast cancer, and it causes resistanceto chemotherapy and radiation [22]. High expression levels ofsurvivin also correlates with an increased rate of tumor recurrenceand resistance to chemo- and radiotherapy [23,24]. So in this studywe use TmSm(T34A) to increase the sensitivity of the cancer cell to

the chemotherapy of doxorubicin. But, not all cell lines weresensitive to the combination treatment from the various results,there were still cell type-specific differences influencing on thefinal synergistic effects with doxorubicin. Therefore based on theprimary test to human breast cancer MCF-7, Bcap-37 and T-47Dcells, the MCF-7 cell lines with most sensitive effect have beendone further research. Under the treatment of the combination ofdoxorubicin with TmSm(T34A), the cell cycle was analyzed by flowcytometry and the cell population had little changed in the G2/Mphase. This indicated TmSm(T34A) might not arrest breast cancercells at the G2/M phase to induce apoptosis to increase doxorubicincytotoxicity.

4.3. TmSm(T34A) playing active role liked to its phosphorylation

defective and other related apoptosis factors

The phosphorylation defective survivin mutant may act as adominant negative mutant for its ability to associate with p34cdc2on the mitotic, which may stimulate caspase-9-depedent apopto-sis. In our study, we checked if TmSm(T34A) induced apoptosisthrough activing intrinsic initiator caspase-9, it was proved asshown in Fig. 4. Phosphorylation of survivin on Thr34 may regulateapoptosis at cell division via an interaction with caspase-9 [25], orloss of survivin phosphorylation on Thr34 resulted in dissociationof a survivin-caspase-9 complex at midbodies and caspase-9-depedent apoptosis at cell division [26]. To further confirm thecaspase pathway, we checked the caspase substrate PARP, a DNArepair protein, in MCF-7 cell lines as a downstream signaling eventindicative of apoptosis. PARP and PARP cleavage were observed aclearly increase in response to doxorubicin and combinedtreatment, which suggests that TmSm(T34A) could improve theapoptotic effect of doxorubicin.

Furthermore, adding TmSm(T34A) also acts on anti-apoptoticproteins Bcl-2, which is associated with the resistance toapoptosis-inducing therapies [4]. Bcl-2 exerts its effect byinteracting indirectly with Bax and preventing caspases activating,such as caspase-9. Overexpression of Bcl-2 is an importantpathway of resistance in treating cancers with chemo- andradiotherapy [27]. Decreasing Bcl-2 can potentially reduceresistance of tumor to chemotherapy. Many preclinical and clinicalstudies suggest that the combination of cytotoxic therapy byantisense oligonucleotide of Bcl-2 results in synergistic anticancereffects in many hematologic and solid tumors, such as breastcancer [28,29]. Therefore, down-regulation of Bcl-2 may be aprincipal strategy for breast cancer therapy. In most diffuse large-cell lymphoma, survivin may act as the node protein of apoptosispathway in a significant subset of patients [30], with another

Y. Xu et al. / Biomedicine & Pharmacotherapy 66 (2012) 368–372372

subset being relied on Bcl-2 overexpression and a smaller distinctpopulation possibly depending on both. TmSm(T34A) can inhibitboth survivin and Bcl-2, which demonstrates its inspiring effect.

4.4. The effect of inducing apoptosis and sensitizing chemotherapy to

breast cancer cells from TmSm(T34A) may be associated and promoted

each other

The mechanism of drug resistance of human breast cancer iscomplex, although there are many explains described, includingexporting drug from the cell, activating detoxifying enzymes,suppressing apoptosis, defects of DNA repair [31] and regulatingepigenetic [32]. But the multiple functions to inducing apoptosis,arresting cell cycles and weakening resistance by TmSm(T34A) tobreast cancer cells were difficult to elucidate from single respect asabove reports. Importantly, the research results suggestedTmSm(T34A) could induce the apoptosis of breast cancer cellsand sensitize chemotherapy not only by down-regulating caspasepathway, but also by interfering survivin function and decreasingBcl-2, both of which associated with the resistance of tumors toapoptosis-inducing therapies. The most likely reason may beTmSm(T34A) plays great roles in multiple pathways in vivobecause the target survivin is nodal protein involved in cellsignaling pathways [33]. Probably, TmSm(T34A) in the studypromises new therapy strategies to stride over these obstacles ofbreast cancer chemotherapy side effects and alleviate harm topatients.

5. Conclusion

This research demonstrates that the combined use ofTmSm(T34A) and doxorubicin, increased the susceptibility ofbreast cancer cells to doxorubicin and efficiently decreased thedosage of doxorubicin. TmSm(T34A) may be a promisingbiotherapeutic drug to breast cancer in combination withchemotherapeutic doxorubicin.

Disclosure of interest

The authors declare that they have no conflicts of interestconcerning this article.

Acknowledgments

This work was supported by the National Natural ScienceFoundation (30873190), the National Science Research Project‘‘Significant New Drugs Created’’ of Eleventh Five-year Plan(2009ZX09103-693) and Key for Fundamental and Interdisciplin-ary Research of Chinese Ministry (WK0913002).

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