Strategy for reversing resistance to a single anticancer agent in human prostate and pancreatic carcinomas

Abstract

Effective therapies for most solid cancers, especially those that have progressed to metastasis, remain elusive because of inherent and acquired resistance of tumor cells to conventional treatments. Additionally, the effective therapeutic window for many protocols can be very narrow, frequently resulting in toxicity. The present study explores an anticancer strategy that effectively eliminates resistant cancer cells without exerting deleterious effects on normal cells. This approach employs melanoma differentiation-induced gene-7/interleukin-24 (mda-7/IL-24), a cancer-specific, apoptosis-inducing cytokine, in combination with nontoxic doses of a chemical compound from the endoperoxide class that decomposes in water generating singlet oxygen. This combinatorial regimen specifically induced in vitro apoptosis in prostate carcinoma cells, with innate resistance to chemotherapy or engineered resistance to mda-7/IL-24, as well as pancreatic carcinoma cells inherently resistant to any treatment modality, including mda-7/IL-24. Apoptosis induction correlated with increased cellular reactive oxygen species production and was prevented by general antioxidants, such as N-acetyl-l-cysteine or Tiron. Induction of apoptosis in combination-treated cancer cells correlated with a reduction in the antiapoptotic protein BCL-x(L). In contrast, both normal prostate and pancreatic epithelial cells were unaffected by the single or combination treatment. These provocative findings suggest that this combinatorial strategy might provide a platform for developing effective treatments for therapy-resistant cancers.

Fig. 1.

Structure of EPX and scheme of EPX synthesis. EPX (1) has a half-life of 25–30 min at 37°C in PBS (39). The thermal decomposition to yield the propionic acid (2) may follow two paths; roughly 45% follows a concertedly path generating 2 and singlet oxygen, whereas the remainder follows a diradical path generating 2 and ground-state oxygen (40, 41).

Fig. 2.

Combination of low-dose EPX (1 μM) with Ad.mda-7 specifically kills cancer but not P69 cells. The killing effect is abrogated by antioxidant (NAC) pretreatment (5 mM). MTT viability assays were performed on day 5 after treatment with the indicated agent(s). Con, no EPX treatment.

Fig. 3.

Combination of low-dose EPX with Ad.mda-7 induces apoptosis selectively in prostate cancer cells. Annexin V binding assays were performed 24 h after treatment with the indicated agent(s). Con, no EPX treatment.

Fig. 4.

MDA-7/IL-24, BCL-x_L, and EF-1α protein expression in prostate cancer and P69 cells after treatment with EPX, Ad.mda-7, or their combination. Cells were treated as indicated, protein lysates were collected 48 h after treatment, and Western blots were performed as described in Materials and Methods.

Fig. 5.

EPX treatment promotes mitochondrial dysfunction and elevated ROS (peroxide and/or nitric oxide) production in cancer but not in P69 cells. Cells were treated (with EPX alone or in combination with Ad.mda-7), stained, and analyzed by flow cytometry. (A) Time course of mitochondrial potential changes in P69 and DU-145 cells upon EPX treatment. (B) ROS production in P69 and prostate cancer cells after Ad.vec + EPX or Ad.mda-7 + EPX treatment. Con, no EPX treatment.

Fig. 6.

Combination of Ad.mda-7 + EPX (0.25 μM) effectively kills pancreatic cancer cells by apoptosis induction and promotes production of MDA-7/IL-24 protein. Pretreatment with Tiron inhibits killing and blocks MDA-7/IL-24 protein production in combination-treated cells. (A) MTT assays performed at day 5. (B) Annexin V binding assays with PANC-1 cells performed 48 h after treatment. (C) ROS production assays with PANC-1 cells performed 24 h after treatment. (D) Western blot for MDA-7/IL-24 protein in PANC-1 and BxPC-3 cells. Con, no EPX treatment.