Synthetic triterpenoids can protect against toxicity without reducing the efficacy of treatment with Carboplatin and Paclitaxel in experimental lung cancer

Abstract

Synthetic oleanane triterpenoids are multifunctional drugs being developed for the prevention and treatment of a variety of chronic diseases driven by inflammation and oxidative stress. Low nanomolar concentrations of triterpenoids inhibit the induction of inflammatory cytokines, and these drugs are potent activators of the Nrf2 cytoprotective pathway. In contrast, low micromolar concentrations of triterpenoids increased the production of ROS and induced apoptosis in a dose-dependent manner in malignant MCF10 CA1a breast cancer cells. Because cancer cells respond differently to ROS than normal cells, it should be possible to exploit these differences therapeutically. In an experimental model of lung cancer, the triterpenoids activated the Nrf2 pathway, as seen by induction of the cytoprotective enzyme NQO1, and reduced the toxicity of carboplatin and paclitaxel. The induction of the Nrf2 pathway in the lung did not suppress the efficacy of treatment with carboplatin and paclitaxel, as the average tumor burden in the group treated with the combination of CDDO-Me and carboplatin/paclitaxel decreased by 90% (P < 0.05 vs. the controls and both single treatment groups). Understanding the dose response of triterpenoids and related drugs will help provide the proper context for optimizing their potential clinical utility.

Keywords: CDDO-Methyl ester; Nrf2; carboplatin toxicity; lung cancer; reactive oxygen species; triterpenoid.

FIG. 1.

The progressive MCF10 model of breast cancer. The MCF10A spontaneously immortalized cell line was derived from non-malignant human breast epithelium and does not grow in immunodeficient mice. The introduction of an activating Ha-Ras mutation into MCF10A cells resulted in a new cell line (AT1) that forms palpable premalignant lesions when injected in vivo. Repeated passing of MCF10 AT1 premalignant lesions into nude mice created highly tumorigenic and invasive MCF10 CA1a cells. These 3 cells lines thus represent a spectrum of progression from the relatively normal MCF10A breast epithelial cells to fully malignant, poorly differentiated MCF10-CA1a breast cancer cells capable of metastasizing.

FIG. 2.

Transformation of MCF10 cells with activated Ras enhances intracellular production of ROS. MCF10A immortalized breast epithelial cells, MCF10 AT-1 premalignant cells containing an activating mutation in H-Ras (A and B) and fully malignant MCF10-CA1a cells (B) were challenged with the oxidant tertbutyl hydroperoxide (tBHP) for 15 min, and reactive oxygen species (ROS) were measured by flow cytometry. These results are representative of 3–4 independent experiments. P < 0.05 vs. control, # P < 0.05 vs. control and vs. MCF10A cells.

FIG. 3.

Different doses of the triterpenoid CDDO-Im reduce or amplify the production of ROS in MCF10 cells. MCF10 A and MCF10 AT-1 cells were treated with CDDO-Im for 16 hrs, incubated with H₂DCFDA for 2 hrs, and challenged with tBHP for 15 min. The fluorescent ROS was detected by flow cytometry, and the results are representative of 3 independent experiments. P < 0.05 vs. control and 0.1 μM CDDO-Im.

FIG. 4.

Higher concentrations of the triterpenoid CDDO-Im alone increase the production of ROS in MCF10 cells. MCF10 AT-1 and CA1a cells were treated with CDDO-Im for 4 hrs, incubated with H₂DCFDA for 1 hr, and ROS were detected by flow cytometry. The results represent 4 independent experiments.

FIG. 5.

The same concentrations of CDDO-Im that increase ROS also induce apoptosis in malignant MCF10 CA1a cells. MCF10 CA1a cells were treated with various concentrations of CDDO-Im for 6 hrs, and annexin V and propidium iodide staining were detected by flow cytometry. The results represent 2 independent experiments.

FIG. 6.

ROS production and apoptosis are higher in Nrf2 knockout fibroblasts than in wild-type cells treated with CDDO-Im. Nrf2^–/– knockout (KO) and wildtype^+/+ (WT) fibroblasts were treated with CDDO-Im or tBHP (250 μM) alone for 4 hrs (A) or were incubated with CDDO-Im for 15 hrs and then challenged with tBHP for 15 min (B) and ROS were detected by flow cytometry. Cells were also treated with CDDO-Im for 14 hr, and the percentage of apoptotic cells determined by annexin V and propidium iodide staining followed by flow cytometry (C). * P < 0.05 vs. control and # P < 0.05 vs. tBHP.

FIG. 7.

Triterpenoids induce NQO1 mRNA (A) and enzyme activity (B) in vivo. Twelve female A/J female mice were fed triterpenoid (CDDO-Me 80 mg/kg diet or CDDO-Ea 800 mg/kg diet) for 1 week. Induction of NQO1 mRNA was measured in peripheral blood mononuclear cells (PBMCs), and NQO1 enzyme activity was analyzed in livers and lungs. * P < 0.05 vs. control.

FIG. 8.

Protocol for lung cancer studies. A. In a pilot study (A), A/J mice were injected with vinyl carbamate (VC). Eight weeks later, the mice were fed triterpenoids in diet. One week later, the mice were injected with 5 doses of carboplatin and paclitaxel (C/P), the first three doses once a week and the last two doses two weeks apart. In a second experiment (B), triterpenoid diets were not started until 12 weeks after initiation with VC, and the 5 C/P injections were given every two weeks.