Inhibition of cisplatin-resistance by RNA interference targeting metallothionein using reducible oligo-peptoplex

    Inhibition of Cisplatin-Resistance by RNA Interference Targeting Metallothionein using Reducible Oligo-Peptoplex Jong-Hwan Lee, Ji-Won Chae, Jang Kyoung Kim, Hyung Jin Kim, Jee Young Chung, Yong-Hee Kim PII: DOI: Reference:

S0168-3659(15)30028-6 doi: 10.1016/j.jconrel.2015.07.015 COREL 7762

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

20 March 2015 23 June 2015 15 July 2015

Please cite this article as: Jong-Hwan Lee, Ji-Won Chae, Jang Kyoung Kim, Hyung Jin Kim, Jee Young Chung, Yong-Hee Kim, Inhibition of Cisplatin-Resistance by RNA Interference Targeting Metallothionein using Reducible Oligo-Peptoplex, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.07.015

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Inhibition of Cisplatin-Resistance by RNA

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Interference Targeting Metallothionein using

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Reducible Oligo-Peptoplex

Jong-Hwan Lee, † Ji-Won Chae, † Jang Kyoung Kim, † Hyung Jin Kim, † Jee Young Chung, † Yong-Hee Kim, ‡,*

Department of Bioengineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu,

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Seoul 133-791, Republic of Korea

Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, 17

Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea

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*Address correspondence to Yong-Hee Kim ([email protected], Tel: +82 2 2220

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ACCEPTED MANUSCRIPT ABSTRACT Effective intracellular level of a platinum anti-cancer drug, cisplatin, following

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repeated injections can be decreased either by the active efflux via ATP pump or by

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interactions with glutathione and metallothionein. Cisplatin in cytoplasm preferably binds to

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cysteine-rich proteins such as glutathione and metallothionein (MT). Detoxification of cisplatin by intracellular thiol-containing proteins has been considered to be major hurdles to overcome. The short hairpin RNA targeting MT (shMT) was tested to down-regulate MT and recover cisplatin resistance. A reducible polymer, poly(oligo-D-arginine) (rPOA), formed

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stable complex with shMT and demonstrated superior transfection efficiency. Efficient transfection of shMT/rPOA oligo-peptoplexes was found to significantly inhibit MT over-

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expression, resulting in 45% decrease of cell viability compared to the cisplatin alone group. This decrease was mediated by synergistic effect of shMT/rPOA oligo-peptoplex and cisplatin. Co-administration of shMT/rPOA oligo-peptoplex and cisplatin in in vivo tumor

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model showed noticeable tumor-suppressing effect by inducing reversal of cisplatin

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resistance following effective intracellular delivery of shMT by rPOA. Combination therapy through co-administration of shMT/rPOA oligo-peptoplex and cisplatin was found to

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effectively reverse cisplatin resistance by RNA interference and consequently improve anti-

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cancer activity of cisplatin.

Keywords. Cisplatin resistance; RNA interference; Metallothionein; Combination therapy

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1. Introduction Cis-diamminedichloroplatinum(II) (Cisplatin, CDDP) was first approved by FDA

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(Food and Drug Administration) in 1978 to treat testicular and bladder cancers. Cisplatin is a

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widely used member of a class of platinum-containing anti-cancer drugs for the treatment of wide spectrum of solid neoplasms including bladder, ovarian, colorectal, lung and head

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cancers [1-3]. Despite the positive anti-cancer effects, cisplatin was reported to induce side effects such as nephrotoxicity, neurotoxicity, myelosuppression, immunosuppression and hearing loss [4, 5]. To reduce the side effects, carboplatin and oxaloplatin as alternative drugs

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were developed. Carboplatin as a second-generation platinum compound reduced nephrotoxicity and neurotoxicity, but it was less potent than cisplatin and failed to treat

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cisplatin-resistant cells. Although oxaloplatin exhibited definite pharmacological and immunological properties compared with cisplatin and carboplatin, it ultimately did not resolve side-effect and showed limitation of anti-tumor efficacy in cisplatin resistant cells.

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Main limitation of cisplatin as an anticancer drug is the chemoresistance. Despite initial

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responses, cisplatin resistance often results in therapeutic failure. An intense research has been conducted on several mechanisms that account for the cisplatin resistance and various

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challenges have been made to overcome [6-8]. Cisplatin exhibits anticancer effects via complex signaling pathway that is intracellularly activated through a series of aquation reactions in cancer cell. One of the

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chloride ligand is replaced by water in cytoplasm through a process called aquation. Aqua complexes of cisplatin are able to bind to N7 of two neighbouring guanine in the same or in opposite DNA strands, which induced cancer cell apoptosis. However, effective intracellular level of cisplatin following repeated injections can be decreased by the active efflux via ATP pump and by interaction with glutathione and metallothionein [9, 10]. The cisplatin resistance resulting from increased inactivation by intracellular proteins has been considered to be major hurdles to overcome. Cisplatin in cytoplasm preferably binds to cysteine-rich proteins such as GSH, methionine and MT [11]. Cisplatin can be covalently linked to GSH following nucleophilic attack of the glutathione thiolate anion. MT, a well-known intracellular antioxidative protein, consists of 61 amino acids including 20 cysteine residues [12]. Cisplatin resistance may be due, in part, to the sequestration of cisplatin or modulation of cisplatin's anti-cancer activity by MT following metal-induced MT over-expression [13, 14]. 3

ACCEPTED MANUSCRIPT It was the aims of current study to confirm the overexpression of MT in cisplatinresistant cells contrary to cisplatin-sensitive cells providing a rationale of our hypothesis that the inhibition of MT expression would enhance the chemo-sensitivity of cisplatin and to

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develop an efficient delivery system consisting of short hairpin RNA against MT (shMT) and

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reducible poly oligo-D-arginine (rPOA) to synergistically improve anti-cancer effect of

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cisplatin (Fig. 1). Previously, we have reported effectiveness and safety of rPOA as a nonviral gene carrier in various in vitro and in vivo models [15, 16]. Tumor allograft models were prepared by subcutaneously injecting cisplatin-resistant B16F10 cells to C57BL/6 mice.

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Synergistic anti-cancer efficacy of shMT/rPOA oligo-peptoplex and cisplatin combination

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therapy was verified in cisplatin-resistant cell line and in vivo mouse cancer model.

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ACCEPTED MANUSCRIPT Fig. 1. Schematic mechanism of reversal of cisplatin resistance by shMT delivery in B16F10 melanoma cells

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2. Materials and Methods

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2.1. Materials

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Metallothionein shRNA plasmid was constructed by Santa Cruz Biotechnology (Santa Cruz, CA). Plasmid consisted of 5 target-specific lenti-viral vectors, each encoding 19-25bp shRNA, which were constructed to inhibit translational process of metallothionein.

CCACAUCUGUGUAAAUAGAtt-3’,

5’-

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Five sequences of sense strand were as follows: 5’-GUUCCACCCUGUUUACUAAtt-3’, 5’CAAG-GACUGUGUGUGCAAAtt-3’,

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5’-CAGCGA-AGUUAAAUCUCAUtt-3’.

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GGAGAUAAUUGCAAAUGCAtt-3’

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Branched PEI (Mw 25 kDa), ampicillin and cisplatin were purchased from Sigma-Aldrich (St. Louis, MO). Plasmid luciferase (pLuc, pGL3-promoter, 5,010 bp) and luciferase assay kit

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were purchased from Promega (Madison, WI). AnnexinV-Phycoerythrin (PE) apoptosis

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detection kit was obtained from BD Pharmingen (San Diego, CA). Cy5-intracellular nucleic acid localization kit was obtained from Mirus Bio Corporation (Madison, WI) and Alexa488

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micro scale labeling kit was purchased from Invitrogen (La Jolla, CA). All other reagents of analytical grade were used without further modification.

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2.2. Cell culture and development of cisplatin resistant cell lines Mouse melanoma cancer cell line (B16F10) was purchased from Caliper Life Sciences (Hopkinton, MA). DMEM (high glucose) containing 10% fetal bovine serum and 1% penicillin were used to cultivate cells at 37℃ with 5% CO2. Cisplatin-resistant cell lines were seeded in cell culture flask at 5 x 104 cells/well. Culture media containing cisplatin at the concentration of 4 μg/ml were added and replaced every other day. Cells survived in the medium containing cisplatin were transferred into the new flask and developed to be resistant by continuous cisplatin exposure for at least 14 weeks. Cisplatin-resistance was confirmed by detecting the cell viability using MTT assay. To confirm the overexpression of metallothionein in cisplatin-resistant cells, cisplatin-sensitive cells and resistant cells were seeded in 12-well plates at a total density of 2 5

ACCEPTED MANUSCRIPT x 105 cells/well. After 24 hours, cells were collected and lysed with 1X lysis buffer (Promega, Madison, WI) on ice. Western blot assay was performed with an anti-metallothionein antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Antibody complexes were detected by

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measuring chemi-luminescence using the ECL kit (GE Healthcare, Milwaukee, MI). A

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protein assay kit (Bio-Rad, Hercules, CA) was used to measure protein concentrations. Anti-β

2.3. Preparation of shRNA/rPOA oligo-peptoplexes

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actin antibody (Cell Signaling, Danvers, MA) was used for western blot control.

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The reducible poly(oligo-D-arginine) (rPOA) was prepared following previously published procedure [15]. In short, Cys-(D-R9)-Cys was incubated in PBS (pH 7.4)

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containing 30% dimethyl sulfoxide at a final concentration of 57 mM. After 6 days, the reaction was terminated by adding 5 mM HEPES buffer. Unreacted low molecular weight peptides were eliminated by dialysis with a membrane of MWCO 10,000 Da. Purified

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peptides were lyophilized using a vacuum-freeze dryer (Freezone 4.5; Labconco, Kansas City,

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MO). shRNA/rPOA oligo-peptoplexes were prepared as we previously reported.[15] Briefly, shRNA and rPOA were dissolved separately in DMEM without fetal bovine serum and

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penicillin. Two solutions were mixed and incubated for 20 minutes at room temperature.

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2.4. Characterization of shMT/rPOA oligo-peptoplexes The rPOA and shRNA oligo-peptoplexes were characterized by measuring size, zeta potential and agarose gel retardation. The surface zeta potential and mean diameter of the oligo-peptoplexes at varying weight ratios were measured by Zeta-sizer Nano ZS (Malvern Instruments Worcestershire, UK). Agarose gel electrophoresis assay was conducted with mixtures of various amounts of rPOA and 1 μg of shRNA incubated for 20 minutes at room temperature. Samples were electrophoresed in 0.8% (w/v) agarose gel for 20 minutes at 100V in 0.5% TBE buffer. All data with shRNA/rPOA oligo-peptoplexes were expressed as weight ratios. Morphology of oligo-peptoplexes was also confirmed by the energy filtered transmission electron microscopy (EF-TEM) (EM9120, Carl Zeiss Vision GmbH, Aalen, 6

ACCEPTED MANUSCRIPT Germany) at the Korea Basic Science Institute (Chuncheon, Korea). Samples were placed on

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TEM grids, dried at 50℃ and coated with uranyl acetate for negative staining.

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2.5. In vitro transfection efficiency

Cisplatin-sensitive and cisplatin-resistant B16F10 cells were seeded on 12-well

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plates at a density of 2.0 × 104 cells/well. After 24 hours incubation, culture medium was replaced with 500 ml of fresh medium. Medium contained 1 μg of pLuc with PEI or rPOA at a weight ratio of 1:2. After 6 hours incubation, 500 ml of normal growth medium containing

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double concentrations of serum and antibiotic were added. After 48 hours, cells were washed two times with PBS and treated with 200 μl of 1X lysis buffer reagents (Promega, Madison,

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WI) in each well. After cell lysates were scraped from each well, they were collected into 1.5 ml microtubes and centrifuged at 13,000 rpm for 3 minutes. The RLU values of luciferase in cell lysates were quantified using 96-well plate luminometer (Berhold Detection System,

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Pforzheim, Germany). Data were expressed as RLU per mg of cell protein measured by the

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DC protein assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin

2.6. MTT assay

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standards dissolved in PBS.

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Anti-tumor synergistic effects of oligo-peptoplexes with cisplatin was confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cisplatinsensitive and cisplatin-resistant cells were seeded in 12-well plates at a density of 2.0 × 104 cells/well. After 24 hours, media were replaced with 500 μl of media without fetal bovine serum and penicillin and cells were treated with 1 μg of shMT/rPOA oligo-peptoplexes at a weight ratio of 1:2. After 6 hours, 500 μl of medium containing double concentration of the normal serum and antibiotic with 25 μg/ml cisplatin were added into cells for additional 24 hours. Relative cell viabilities were measured three times with three replicates and expressed as percent cell viabilities.

2.7. Cellular uptake 7

ACCEPTED MANUSCRIPT The cellular uptake of shMT delivered by rPOA was examined by confocal microscopy. B16F10 cisplatin-resistant cells were seeded in 12-well plates at a density of 2 x 104 cells/well for 24 hours before transfection. The oligo-peptoplexes containing 1 μg of

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Cy5-shMT and 2 μg of Alexa488-rPOA were incubated with B16F10 melanoma cells for 3

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hours and 6 hours. Subsequently, the distribution of shMT/rPOA oligo-peptoplexes in cells

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was examined using confocal laser scanning microscopy (CLSM, LSM510 META NLO; Carl Zeiss Jena GmbH, Germany) at the Korea Basic Science Institute (Chuncheon, Korea).

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2.8. MT silencing effect of shMT/rPOA oligo-peptoplex

In order to evaluate the down-regulation of MT expression following intracellular

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delivery of the shMT/rPOA oligo-peptoplexes, the western blot analysis with a MT-specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was performed. Cisplatin-resistant

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cells were seeded in 12-well plates at a density of 2.0 × 104 cells/well. After 24 hours, media were replaced with fresh media containing 1 μg of shMT/rPOA oligo-peptoplexes at a weight

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ratio of 1:2. After 6 hours, 500 μl of media containing double concentration of the normal serum and antibiotic were added to cells. After 12, 24, 48 hours of incubation, cells were

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washed 2 times and lysed with 60 μl of 1X lysis buffer. Cell lysates were evaluated using

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western blot analysis.

2.9. Apoptosis evaluation by flow cytometry A AnnexinV-Phycoerythrin(PE) detection kit (BD PharMingen, San Diego, CA) was used for the analysis of apoptosis. B16F10 cells were seeded on the 12-well plates at a density of 2.0 × 104 cells. After 24 hours incubation, 1 μg of naked shMT or shMT/rPOA oligo-peptoplexes at a weight ratio of 1:2 in 500 μl of medium without fetal bovine serum and penicillin were added. After 6 hours, 500 μl of medium containing 25 μg/ml cisplatin were added into cells followed by additional 20 hours incubation. Then, cells were harvested and washed twice with cold PBS and suspended in 500 μl of binding buffer. Cells were reacted with PE-conjugated AnnexinV and 7-amino-actinomycin D (7-AAD) at room temperature for 15 minutes. Stained cells were analyzed using a BD FACSCalibur flow 8

ACCEPTED MANUSCRIPT cytometer (BD Bioscience, San Jose, CA). Total 10,000 events per each group were counted and evaluated. Each experimental point was performed in triplicate and data analysis was

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performed by BD CellQuest software (BD Biosciences, Franklin Lakes, NJ,).

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2.10. In vivo anti-tumor effects

B16F10 melanoma cells were injected into six week old male C57BL/6 mice. Prior to injection, cisplatin-sensitive and cisplatin-resistant cells were cultivated at 37℃ with 5%

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CO2. Then, 1.0 × 106 cells were suspended in 100 μl PBS and injected subcutaneously into right hind limb of the mice. Transplanted mice were raised for 1 week for optimum tumor

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growth.

Tumor volume and body weight were measured for the evaluation of anti-tumor efficacy and toxicity. Tumor size was measured from the longest (a) and shortest (b)

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diameters using vernier calipers, and tumor volume was calculated using an equation of

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V=0.5ab2. When tumor diameter reached ~ 100 mm3, mice were grouped (n=5) and cisplatin and shMT/rPOA oligo-peptoplexes were injected. Cisplatin (3 mg/kg) was intraperitoneally injected. The shMT/rPOA oligo-peptoplexes were prepared by reacting 3 μg of shMT and 6

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μg of rPOA at a weight ratio of 1:2 in 50 μl PBS. Along with intraperitoneal injections of cisplatin, shMT/rPOA oligo-peptoplexes were locally injected five times into mice. The body

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weights of mice were measured every other day.

2.11. In vivo metallothionein level Total metallothionein level in tumor tissues were observed after administration of cisplatin, shMT/rPOA oligopeptoplexes. Tumor tissues were dissected, and equal weights were measured. Equal amount of tumor tissues were homogenized in lysis buffer using a homogenizer and lysed by freeze-thaw cycle and was centrifuged at 13,000rpm for 15 minutes. The total metallothionein protein level was quantified using the supernatants by western blot analysis using a metallothionein-specific antibody (Santa Cruz Biotechnology).

3. Results and Discussion 9

ACCEPTED MANUSCRIPT 3.1. Development of cisplatin resistance in a B16F10 melanoma cell line Drug resistance was induced by continuous exposure of B16F10 malignant cancer

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cells to cisplatin for 14 weeks. Cells were initially treated with cisplatin at the initial

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concentration of 1 μg/ml and gradual increase up to 4 μg/ml. To confirm cisplatin resistance, MTT assay was conducted on cisplatin-sensitive and cisplatin-resistant cells 24 hours after

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treatment with varying cisplatin concentrations. Cisplatin-resistant cells showed higher cell viability than cisplatin-sensitive cells, maintaining viability of approximately 80% at 50 μg/ml (Fig. 2A). It was also found that as the exposure time of cells to cisplatin increased, the

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resistance increased.

Platinum-based anti-cancer drugs are inactivated by glutathione (GSH) or

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metallothionein (MT) possessing thiol functional groups. Thiol-containing molecules play the roles of metal homeostasis and detoxification by formation of redox environments. MT has been known to be overexpressed in cisplatin-resistant cells and induce resistance against

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cisplatin by detoxification [17]. The western blot result demonstrated overexpression of MT

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in B16F10 cisplatin-resistant cells contrary to cisplatin-sensitive cells (Fig. 2B), which provided a rationale of our hypothesis that the inhibition of MT expression would enhance

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the chemo-sensitivity of cisplatin.

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Fig. 2. Development of cisplatin-resistance in a B16F10 melanoma cell line (A) Cell viability of cisplatin-sensitive cells and cisplatin-resistant cells at various concentration of cisplatin from 0 μg/ml to 50 μg/ml. (B) Expression levels of metallothionein in cisplatin-sensitive cells and cisplatin-resistant cells verified by western blot. β-actin levels indicate equal amounts of protein (40 μg) in both groups. 10

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3.2. Preparation and characterization of shMT/rPOA oligo-peptoplexes

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In previous studies, we developed reducible poly(oligo-D-arginine) (rPOA), a non-

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viral vector displaying remarkable transfection efficiency and low toxicity, which is efficient

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for the delivery of DNA and siRNA to treat diverse diseases [15, 18]. The rPOA was synthesized via DMSO-mediated oxidative polymerization of Cys-(D-9R)-Cys oligo-peptides. To verify optimum ratios between shMT and rPOA, shMT/rPOA oligo-peptoplexes were characterized by measuring diameter and zeta-potential using dynamic light scattering (DLS).

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The shMT/rPOA oligo-peptoplexes at a weight ratio of 1:2 formed spherical complexes of 160 nm average diameter, while those of 1:1 weight ratio showed average diameter of 700

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nm (Fig. 3A). Although zeta-potentials were positive at both ratios (Fig. 3B), the shMT/rPOA oligo-peptoplexes at a weight ratio of 1:2 were more capable of condensing shMT into nanosized spherical aggregates with fine poly-dispersity indexes (PDI).

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To test condensation efficiency, the gel retardation assay was conducted with

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shMT/rPOA oligo-peptoplexes at weight ratios of 1:0.5, 1:1 and 1:2 (Fig. 3C). Ionic interaction between shMT and rPOA resulted in complete retardation of oligo-peptoplex at a

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weight ratio of 1:2.

The morphology of shMT/rPOA oligo-peptoplexes at a weight ratio of 1:2 was examined by transmission electron microscopy (TEM) (Fig. 3D). TEM image of oligo-

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peptoplexes showed nanometer-sized (100-200 nm) spherical particles with a variation of size. These results indicated that shMT/rPOA oligo-peptoplexes at weight ratio of 1:2 were suitable for further studies.

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Fig. 3. Characterization of shMT/rPOA oligo-peptoplexes (A) Diameter and (B) zeta potential of shMT/rPOA oligo-peptoplexes at weight ratios of 1:1 and 1:2. The rPOA was mixed with 1 μg of shMT to make oligo-peptoplex by incubating 20 minutes in PBS. (C)

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Condensation of shMT/rPOA oligo-peptoplexes were confirmed by agarose gel

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electrophoresis. (D) TEM images of shMT/rPOA oligo-peptoplexes. x31,200 (Scale bar = 1

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μm)

3.3. In vitro transfection and cytotoxicity of shMT/rPOA oligo-peptoplex To verify transfection efficiency of rPOA in B16F10 melanoma cells, luciferase

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assay was conducted using luminometer. The luciferase plasmid (pLuc) amount was fixed at 1 μg and the weight ratio of pLuc and PEI or POA was 1:2 (Fig. 4A). The pLuc/rPOA oligopeptoplex showed significantly better transgene expression than pLuc/PEI oligo-peptoplex in both cisplatin-sensitive cells and cisplatin-resistant cells. Biocompatibility of oligo-peptoplexes is an important factor for practical application. Cytotoxicity was tested with shMT and cationic polymers (PEI and rPOA) in B16F10 melanoma cells (Fig. 4B). Complexes were prepared with 1 μg shMT at a weight ratio of 1:2. The rPOA at various concentrations of 1~4μg were evaluated for cytotoxicity. The shMT/rPOA complex at a weight ratio of 1:2 showed no cytotoxicity in both cisplatinsensitive and cisplatin-resistant cells, while PEI complex provided severe toxicity. The rPOA alone demonstrated no cytotoxicity with 1μg in both cells and 2-3μg in cisplatin-resistant 12

ACCEPTED MANUSCRIPT cells. Slight toxicity was observed especially in sensitive cells at higher concentrations. These results demonstrated that rPOA is an efficient and safe carrier for shRNA-mediated gene delivery.

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The shMT/rPOA oligo-peptoplexes need to be efficiently internalized into cells and

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finally deliver shMT to nucleus. For the purpose of visualizing intracellular trafficking of

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oligo-peptoplex, shMT and rPOA were labeled with red color Cy5 and green color Alexa488 dyes, respectively. Cellular uptake and distribution of shMT/rPOA oligo-peptoplexes were monitored using confocal laser scanning microscopy (CLSM). Cell nuclei were counter-

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stained with DAPI. Efficient cellular uptake of Alexa488-rPOA and Cy5-shMT oligopeptoplexes into cells was observed while shMT alone was not transfected at all. The shMT/rPOA oligo-peptoplex can be diffusive into cytoplasm within 6 hours since then shMT

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is dissociated (Fig. 4C). In this study, plasmid-based shMT was used instead of siMT and the MT silencing effect was measured 48 hours after further incubation following shMT/rPOA treatment with cells for 6 hours. CLSM images demonstrate dissociated shMT (red in nucleus)

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and rPOA (green in cytoplasm) 6 hours after treatment, while co-localization as complexes

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(yellow) in cytoplasm 3 hours after treatment (Fig. 4D).

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Fig. 4. Cellular uptake and intracellular distribution of shMT/rPOA oligo-peptoplexes in

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B16F10 melanoma cancer cells (A) In vitro transfection efficiency 48 hours after treatment with rPOA/pLuc and PEI/pLuc oligo-peptoplexes at the weight ratio of 2 in cisplatinsensitive cells and cisplatin-resistant cells. (B) Cytotoxicity of shMT and cationic polymers (PEI and rPOA) in cisplatin-sensitive cells and cisplatin-resistant cells. Complexes were prepared with 1 μg shMT at the weight ratio of 2. (C) Cellular uptake and distribution of shMT/rPOA oligo-peptoplexes monitored using CLSM for 6 hours. The shMT was labeled with Cy5 dye (red); rPOA was labeled Alexa488 dye (green); cell morphologies were visualized in DIC images (gray); nuclei were counterstained with DAPI (blue) and the images were merged (right). (Scale bar = 50 μm) (D) CLSM images of B16F10 cisplatin-resistant cells incubated with Cy5-shMT (red)/Alexa488-rPOA (green) oligo-peptoplexes for 3 and 6 hours. (Scale bar = 20 μm). 14

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3.4. In vitro enhancement of cisplatin-induced cytotoxicity by shMT/rPOA oligo-peptoplexes To confirm enhancement of cisplatin-induced cytotoxicity by shMT/rPOA oligo-

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peptoplexes in vitro, cell viability was assessed by MTT assay with B16F10 cisplatin-

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sensitive cells and cisplatin-resistant cells (Fig. 5A). Cisplatin-sensitive cells showed around

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30% of cell viability in the presence of cisplatin. There was no significant difference by additional treatment with shMT as expected from the finding of no overexpression of MT in cisplatin-sensitive cells. Unlike cisplatin-sensitive cells, cisplatin-resistant cells maintained

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high cell viabilities despite the treatment with cisplatin. Efficient transfection of shMT/rPOA oligo-peptoplexes was found to significantly inhibit MT over-expression, resulting in 45% decrease of cell viability compared to the cisplatin alone group. This decrease was mediated

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by synergistic effect of shMT/rPOA oligo-peptoplex and cisplatin. Through western blot, down-regulation of MT expression levels by shMT/rPOA oligo-peptoplex delivery was verified in B16F10 cisplatin-resistant cells (Fig. 5B).

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Treatments with shMT/rPOA oligo-peptoplexes for 12-48 hours effectively inhibited

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expression of MT down to the level of cisplatin-sensitive cells. To examine whether the combination therapy with cisplatin and shMT/rPOA oligo-

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peptoplexes could enhance cisplatin-induced apoptosis, apoptosis degrees in cisplatinsensitive cells and cisplatin-resistant cells were measured by flow cytometry. Co-delivery of both shMT/rPOA oligo-peptoplex and cisplatin significantly increased cisplatin-induced

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apoptosis (Fig. 5C). Compared to cisplatin treated group, apoptosis increased double with codelivery. These results represent that overexpression of metallothionein is one of the main causes for cisplatin resistance and provides a rationale for shMT delivery to reduce it. In order to confirm that MT protein is a main cause of inducing cisplatin resistance, a MT fusion protein conjugated with a protein transduction domain, Trans-activator of transcription (Tat; GRKKRRQRRRPQ), was treated to cisplatin-sensitive cells and cisplatinresistant (Fig. 5D). Since cisplatin-sensitive cells did not overexpress MT protein, transduction of exogenous MT protein mediated by Tat increased intracellular level of MT so that cisplatin resistance was induced, resulting in increased cell viabilities compared to those of cisplatin alone and negative control with Tat-GFP. On the other hand, since cisplatinresistant cells already contained high level of overexpressed MT protein, delivery of 15

ACCEPTED MANUSCRIPT exogenous MT protein showed no significant differences compared to controls. These results demonstrate notable evidence that MT protein have an intimate relation with the cisplatin

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resistance in cancer cells.

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Fig. 5. In vitro enhancement of cisplatin-induced cytotoxicity by shMT/rPOA oligopeptoplexes (A) Enhancement of cisplatin-induced cytotoxicity by down regulation of metallothionein expression. Cell viability was assessed by MTT assay with B16F10 cisplatinsensitive cells and cisplatin-resistant cells 24 hours after treatment with cisplatin, cisplatin+naked shMT and cisplatin+shMT/rPOA oligo-peptoplexes. (The data expressed as mean ± S.D. of three times with four replicates. *p < 0.01) (B) Metallothionein silencing effect by delivery of shMT/rPOA oligo-peptoplex. Western blot was conducted at various incubation times and MT levels are compared with that of cisplatin-sensitive cell. (C) Assessment of apoptosis detected by flow cytometry using AnnexinV in cisplatin-resistant cell lines. The data indicated that treatment with cisplatin combined with shRNA against metallothionein led to enhanced apoptosis in cisplatin-resistant cells. (The data expressed as mean ± S.D. of three times independent experiments with four replicates. *p < 0.01) (D) 17

ACCEPTED MANUSCRIPT Effects of intracellular transduction of exogenous MT fusion proteins on the cell resistances and viabilities in B16F10 cisplatin-sensitive cells and cisplatin-resistant cells. Tat-MT and Tat-GFP fusion proteins were treated to cells for 24 hours at 3 μM. Cell viability was

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determined using MTT assay. (The data expressed as mean ± S.D. of three times with four

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replicates. *p < 0.01)

3.5. Enhancement of in vivo anti-tumor effect of cisplatin by co-administration of shMT/rPOA oligo-peptoplex

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Tumor allograft models were prepared by subcutaneously injecting cisplatin-resistant B16F10 cells to C57BL/6 mice. To investigate anti-cancer efficacy and toxicity, average

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tumor volume (Fig. 6B) and body weight (Fig. 6C) were measured. Cisplatin only treated group showed meager tumor inhibitory effect that resulted from cisplatin resistance of implanted B16F10 melanoma cells. Furthermore, co-delivery of naked shMT and cisplatin

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demonstrated similar effect with cisplatin only treated group. On the contrary, co-

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administration of shMT/rPOA oligo-peptoplexes and cisplatin showed noticeable tumorsuppressing effect by inducing reversal of cisplatin resistance following effective intracellular

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delivery of shMT by rPOA. Along with intraperitoneal injections of cisplatin, shMT/rPOA oligo-peptoplexes were locally injected five times into mice and the average tumor volumes at 12 days were measured to be ~1934 mm3, ~1365 mm3, ~1294 mm3 and ~836 mm3 with control, cisplatin alone, cisplatin (3 mg/kg) with naked shMT (shMT 3 μg), and cisplatin with

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shMT/rPOA oligo-peptoplexes (shMT 3 μg) groups, respectively (p < 0.05). The body weights of mice were measured every other day and remained steady without toxicity in all groups (Fig. 6C). In addition, intratumoral metallothionein levels measured following five local injections demonstrated that the shMT/rPOA + cisplatin groups showed downregulation of metallothionein level compared to those of cisplatin or naked shMT + cisplatin groups as shown in Fig. 6A. Through in vivo experiments using tumor model of C57BL/6 mice, tumor volume was suppressed with shMT/rPOA oligo-peptoplexes resulting in increased sensitivity to cisplatin. These results suggest that therapeutic effect was markedly improved by shMT/rPOA oligo-peptoplexes, which leads to intracellular transfection of cisplatin and noticeable decrease of MT expression in cisplatin-resistant cancer cell. 18

Co-delivery of

ACCEPTED MANUSCRIPT shMT/rPOA oligo-peptoplexes and cisplatin showed synergistic enhancement in the anti-

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cancer efficacy.

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Fig. 6. In vivo anti-tumor effect and toxicity of the combination therapy with shMT/rPOA oligo-peptoplexes and cisplatin (A) Metallothionein silencing effect by delivery of

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shMT/rPOA oligo-peptoplex. Western blot was conducted at various incubation times and MT levels were compared with shMT+cisplatin, cisplatin alone and control groups (B) tumor growth rate and (C) body weight change. (The data represent the average ± S.E. of five mice. *p < 0.05)

4. Conclusions A reducible gene carrier, rPOA, was prepared by DMSO-mediated oxidative method using Cys-(D-9R)-Cys oligomer as a repeat unit for delivery of shRNA targeting MT. The shMT/rPOA oligo-peptoplexes at a weight ratio of 1:2 were formed stable nano-sized (100200 nm) spherical complexes with net positive charge and homogeneous distribution. 19

ACCEPTED MANUSCRIPT Efficient transfection of shMT/rPOA oligo-peptoplexes was found to significantly inhibit MT over-expression, resulting in 45% decrease of cell viability compared to the cisplatin alone group. This decrease was mediated by synergistic effect of shMT/rPOA oligo-peptoplex and

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cisplatin. Co-administration of shMT/rPOA oligo-peptoplex and cisplatin in in vivo tumor

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model showed noticeable tumor-suppressing effect by inducing reversal of cisplatin

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resistance following effective intracellular delivery of shMT by rPOA. This study provides a

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potential remedy of inhibiting platinum drug resistance by the RNA interference method.

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Acknowledgments

This work was partially supported by grants from the National Research Foundation of Korea (2014049587), the Brain Korea 21 plus program (22A20130011095), and the

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Korean Health Technology R&D project through the Ministry of Health &Welfare (HI13C-

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1938-010014).

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