Combination of autophagy inducer rapamycin and oncolytic adenovirus improves antitumor effect in cancer cells - PubMed (original) (raw)

Combination of autophagy inducer rapamycin and oncolytic adenovirus improves antitumor effect in cancer cells

Pei-Hsin Cheng et al. Virol J. 2013.

Abstract

Background: Combination of oncolytic adenoviruses (Ads) and chemotherapy drugs has shown promising therapeutic results and is considered as a potential approach for cancer therapy. We previously have shown that autophagy may generate decomposed cellular molecules that can be used as nutrition to support virus replication in cancer cells. In this study, we evaluated a unique combination of the novel oncolytic Ad-cycE with rapamycin, an autophagy inducer and first-line chemotherapeutic drug.

Methods: The combination of oncolytic Ad-cycE and the autophagy inducer rapamycin was assessed for enhanced antitumor effect. We also evaluated the combined effects of rapamycin and Ad-cycE on cancer cell viability. The interaction between Ad-cycE and rapamycin was analyzed with Calcusyn (Biosoft, Ferguson, MO).

Results: We show that rapamycin induces autophagy, enhances Ad E1A expression and increases Ad oncolytic replication. Combination of rapamycin and Ad-cycE elicits stronger cytotoxicity than single treatment alone. The analyzed data indicates that the Ad-cycE and rapamycin combination has a significantly synergistic antitumor effect.

Conclusions: Our study provides a new insight into vector development and demonstrates the novel roles of autophagy in adenovirus replication. The combination of autophagy-induced chemotherapy and oncolytic virotherapy may be a new approach to improve future cancer treatment.

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Figures

Figure 1

Figure 1

Structure of the Ads. The wild-type Ad (Adwt) with the E1a and E1b genes and their endogenous promoters is shown at the top. The left inverted terminal repeat (ITR), the promoters for E1a gene and E1b genes (E1a-P and E1b-P) and the E1a and E1b open reading frames are indicated. The solid lines represent Adwt regions in these viruses, and the dashed lines represent the deleted regions. AdGFP contains the complete deletion of E1a and E1b regions and their promoters. Ad-cycE contains the deletion of E1b region and a cyclin E promoter (cycE-P) was inserted to replace the deleted E1a promoter.

Figure 2

Figure 2

Selective oncolytic replication of Ad-cycE. (A) WI-38, MCF10A, A549, H1299 and MDA-MB-231 cells were infected with AdGFP, Adwt, or Ad-cycE at 5 MOI. CPE was observed at 48 hr or 72 hr p.i and photographed with an inverted microscope Olympus CKX41. (B) Viral yields produced in WI-38, H1299 and A549 cells were determined at 72 hr p.i. with the infection unit method. The values represent the means ± S.D. of independent triplicate. * P<0.05, Student’s _t_-test. (C) A549 cells were co-infected with 5 MOI AdGFP and mock-infection, AdlacZ, Adwt or Ad-cycE. All fluorescent microscopy is taken at day 1 and day 2 p.i. with an Olympus IX50 microscope (original magnification of ×100).

Figure 3

Figure 3

Effects of rapamycin on cytotoxicity and autophagy. A549 cells were treated with 0 nM, 100 nM, and 200 nM ramamycin (LC Laboratories, Woburn, MA) and collected at 24 hr after treatment. (A) Cell lysates were immunoblotted for LC3 and actin. Actin was used as a loading control. The values indicate the ratios of normalized band intensities of LC3-II to LC3-I. (B) A549 cells were treated with 0 nM, 100 nM, 200 nM, 400 nM, 600 nM and 800 nM ramamycin. The cell viability % was determined at 72 hr after treatment. The values of cell viability % represent the means ± S.D. of independent quadruplicate compared with the 0 nM-control group.

Figure 4

Figure 4

Effects of combination of rapamycin and Ad-cycE on A549 cells. A549 cells were non-treated or treated with 200 nM rapamycin alone, 0.5 or 1 MOI Ad-cycE alone, or the combination treatment of 200 nM rapamycin and 0.5 or 1 MOI Ad-cycE. (A) The results were quantitated into cell viability %. The values of cell viability % represent the means ± S.D. of independent triplicate compared with the mock-control group. * P<0.05, Student’s _t_-test. (B) CPE was photographed at a magnification of x100 at 48 hr p.i.

Figure 5

Figure 5

Effects of rapamycin on the oncolytic replication of Ad-cycE. A549 cells were non-treated or treated with 200 nM rapamycin alone, 0.5 MOI Ad-cycE alone, combination treatment of 200 nM rapamycin and 0.5 MOI Ad-cycE. (A) Viral yields were determined at 48 hr p.i. with the infection unit method. The values represent the means ± S.D. of independent triplicate. (B) Cell lysates harvested at 18 hr p.i. were immunoblotted for Ad E1A and actin. Actin was used as a loading control.

Figure 6

Figure 6

Analysis by Calcusyn (Biosoft, Ferguson, MO) of the interaction between rapamycin and Ad-cycE on A549 cells. Cells were treated with rapamycin alone, Ad-cycE alone or combination of both for 96 hr. (A) The results were quantitated into cell viability %. The values of cell viability % represent the means ± S.D. of independent triplicate compared with the mock-control group. (B) The quantitated cell viability data were analyzed by CalcuSyn software. The X-marks represent the combination index (CI) values of the combination treatment groups. The middle curve line represents the simulated combination index values of the combination treatment groups surrounded by two lines of algebraic estimations of the 95% confidence intervals. (C) The effective concentration EC50, EC75 and EC90 refer to the concentration of a drug or combination of drugs that induces 50%, 75% and 90% inhibition of cell viability. In the conservative isobologram plot, the three curves of the expected EC50, EC75 and EC90 additive effect lines for the combination treatments are labeled; the individual points of EC50, EC75 and EC90 for the combination treatments were indicted by arrows and located below their additive interaction lines, respectively.

Figure 7

Figure 7

Effects of combination of rapamycin and Ad-cycE on MDA-MB-231 cells. (A) Cells were treated with rapamycin alone, Ad-cycE alone or combination of both for 96 hr. The results were quantitated into cell viability %. The values of cell viability % represent the means ± S.D. of independent triplicate compared with the mock-control group. (B) The quantitated cell viability data were analyzed by CalcuSyn software. In the fraction affected versus combination index (Fa-CI) plot, all experimental CI values at the tested ratio were significantly < 1 and within the confidence lines.

Figure 8

Figure 8

Effects of combination of rapamycin and wild-type Ad on A549 cells. (A) Cells were treated with rapamycin alone, Adwt alone or combination of both for 72 hr. The results were quantitated into cell viability %. The values of cell viability % represent the means ± S.D. of independent triplicate compared with the mock-control group. (B) The quantitated cell viability data were analyzed by CalcuSyn software. In the fraction affected versus combination index (Fa-CI) plot, all experimental CI values at the tested ratio were significantly < 1 and within the confidence lines.

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