Novel anti-cancer drug COTI-2 synergizes with therapeutic agents and does not induce resistance or exhibit cross-resistance in human cancer cell lines

Abstract

Emerging drug-resistance and drug-associated toxicities are two major factors limiting successful cancer therapy. Combinations of chemotherapeutic drugs have been used in the clinic to improve patient outcome. However, cancer cells can acquire resistance to drugs, alone or in combination. Resistant tumors can also exhibit cross-resistance to other chemotherapeutic agents, resulting in sub-optimal treatment and/or treatment failure. Therefore, developing novel oncology drugs that induce no or little acquired resistance and with a favorable safety profile is essential. We show here that combining COTI-2, a novel clinical stage agent, with multiple chemotherapeutic and targeted agents enhances the activity of these drugs in vitro and in vivo. Importantly, no overt toxicity was observed in the combination treatment groups in vivo. Furthermore, unlike the tested chemotherapeutic drugs, cancer cells did not develop resistance to COTI-2. Finally, some chemo-resistant tumor cell lines only showed mild cross-resistance to COTI-2 while most remained sensitive to it.

Conflict of interest statement

Competing Interests: JK and SMV have no competing interests with respect to patents, products in development, marketed products, or other matters pertaining to Critical Outcome Technologies Inc. (COTI) (now known as Cotinga Pharmaceuticals, Inc.), its products, or its products in development. JK is the recipient of funding from COTI to conduct the studies reported in this manuscript, without restrictions of any kind on freedom to publish results (other than reasonable advance notice to COTI on the nature of those results). JK received COTI-2 (the compound assessed and reported on in this manuscript) free-of-charge from COTI. Neither JK nor SMV are employed by COTI or Cotinga Pharmaceuticals, Inc. in any capacity whatsoever. Neither JK nor SMV receive any payment (in cash or in-kind) from COTI or Cotinga Pharmaceuticals, Inc. for consulting, nor have they received payments for consulting in the past. KYS is a paid employee of COTI (now known as Cotinga Pharmaceuticals, Inc.) and WRD is a former paid employee of COTI. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. COTI-2 enhances the cytotoxic activity of paclitaxel and cisplatin.

DMS-114 (A and C) and SHP-77 cells (B and D) were cultured overnight then exposed to the indicated doses of paclitaxel and cisplatin plus or minus a pre-determined dose of COTI-2 (IC25) for 4 days before cell viability was determined. The asterix (*) indicates a significant greater-than-additive effect in the combination therapy compared to single agent alone, p<0.05, Student’s t-test. Data are the average mean of 3 independent experiments ± SEM. (E) AN3-CA human endometrial cells (1 x 107) were injected into the right flanks of athymic nude mice (n = 10 mice per group). Xenografts were grown to an average volume of 170 mm3 before animals received treatment i.v. Vehicle control and COTI-2 (25 mg/kg) were administered 3 times a week on alternate days until study end. The schedule for paclitaxel was daily for 5 days (5 mg/kg). In the combination arm, animals received COTI-2 (25 mg/kg) 3 times a week for the entire study and 5 injections of paclitaxel (5 mg/kg). *Significantly different from the paclitaxel alone treatment group, Student’s t-test, p<0.05. Error bars represent SEM.

Fig 2. Only some chemotherapeutic drugs with similar molecular targets show enhanced activity when combined with COTI-2.

DMS-114 (A and C) and SHP-77 (B and D) SCLC cells were treated with various concentrations of carboplatin (A and B) or vincristine (C and D) in combination with or without an IC25 concentration of COTI-2 for 4 days before cell viability was determined. The asterix (*) indicates a significant greater-than-additive effect in the combination therapy compared to single agent alone, p<0.05, Student’s t-test. Data are the average mean of 3 independent experiments ± SEM. (E and F) PANC-1 human pancreatic carcinoma cells (2 x 106) were injected into each flank of NCr-nu mice (n = 12 mice per group). Xenografts were grown to ~100 mm3 before animals received treatment, which consisted of the vehicle control, COTI-2 (125 mg/kg), gemcitabine (100 mg/kg), or the combination (COTI-2 at 125 mg/kg and gemcitabine at 100 mg/kg) (E) or the vehicle control, COTI-2 (125 mg/kg), abraxane (15 mg/kg), or the combination (COTI-2 at 125 mg/kg and abraxane at 15 mg/kg) (F). COTI-2 was delivered p.o. with a schedule of 5 days on treatment and 2 days off weekly. Gemcitabine (100 mg/kg) was administered i.p., every second day, for a total of 6 injections. Abraxane (15 mg/kg) was administered i.v., once per day for 5 consecutive days. The dosing schedule for the combination treatments was identical to that of the single agent treatments for each drug. COTI-2 administration was initiated 1 day after treatment with either gemcitabine or abraxane. *Significantly different from single agent gemcitabine or abraxane treatment groups, Student’s t-test, p<0.05.

Fig 3. The effect of COTI-2 treatment on U87-MG cells in combination with temsirolimus and rapamycin.

U87-MG human glioma cells were cultured in the presence of various concentrations of temsirolimus plus COTI-2 (A) or rapamycin plus COTI-2 (B) for 4 days before cell viability was determined. Black circles indicate the combination of COTI-2 and temsirolimus (A) or rapamycin (B) and the white circles indicate treatment with COTI-2 alone. Data are the average mean of 6 independent experiments ± SEM.*Significant difference, Student’s t-test, p<0.05.

Fig 4. Combining COTI-2 with cetuximab and erlotinib synergistically enhances the efficacy of these drugs against human colorectal cancer cells.

Human colorectal cancer cell lines HCT-15 (A), SW-620 (B), and COLO-205 (C) were treated with varying concentrations of COTI-2, cetuximab, erlotinib, or a combination of COTI-2 and either EGFR inhibitor. Tumor cells were allowed to proliferate for 4 days in the presence of drug(s) before cell viability was determined. All data points indicate the mean of 5 independent measures of viability ± SEM. *Significant difference from cells treated with COTI-2 alone using a Student’s t-test (p<0.05).

Fig 5. Cancer cells do not develop acquired resistance to COTI-2 unlike treatment with paclitaxel and cisplatin.

A549 NSCLC (A), DMS-153 SCLC (B) and SHP-77 SCLC (C) cells were cultured in IC50 concentrations of COTI-2, paclitaxel, or cisplatin for 4 rounds of treatment (5 generations of cells including the parental cells). The surviving 50% of cells from the initial IC50 tested were harvested and cultured for 5 days, after which time this new generation of cells was re-treated with the same agent and a new IC50 value was established. Emerging resistance was identified by increasing IC50 values in successive generations. Significant differences were assessed by Student’s t-test (p<0.05). *Significantly different from parental cells treated with the mentioned drug. Data points indicate the mean from 3 independent experiments ± SEM.

Fig 6. Chemo-resistant cancer cell lines often do not show cross-resistance to COTI-2.

Paclitaxel-resistant (A) and cisplatin-resistant (B) A549, DMS-153, and SHP-77 cells were exposed to IC50 concentrations of COTI-2 and tumor cell proliferation was measured after approximately 4 doublings of control cells. 5FUdR-resistant HeLa cells (C) and vincristine-resistant HN-5a cells (D) were exposed to IC50 concentrations of COTI-2 as described in (A) and (B). Significant differences were assessed by Student’s t-test (p<0.05). Data indicates mean values derived from 3 independent experiments ± SEM.