Bevacizumab and CCR2 Inhibitor Nanoparticles Induce Cytotoxicity-Mediated Apoptosis in Doxorubicin-Treated Hepatic and Non-Small Lung Cancer Cells

Abstract

Non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC) are very common in certain population around the world. Despite the recent advances in their diagnosis and therapy, their prognosis remains poor due to the development resistance to drug. Although doxorubicin (DOX) is considered to be one of the most anti-solid tumor drugs, developed resistance is contributing to unsuccessful outcome. The rationale of the current study is to explore the sensitizing capability of the DOX-treated cancer cells using the anticancer agents; bevacizumab (avastin; AV) and CCR2 inhibitor (CR) in their free- and nano-formulations. Here, the average size, polydispersity index (PDI), zeta potential, and entrpment effeciency (EE%) of the synthesized nanoparticles were measured. We investigated the effect of these platforms on the proliferation, apoptosis, necrosis, nitric oxide (NO), malondialdehyde (MDA), and zinc levels of human HCC (HepG2 and Huh-7) and NSCLC (A549) cancer cell lines. Glucose consumption rates using Huh-7 and A549 cancer cells were tested upon treatments. We demonstrated that AV and CR nano-treatments significantly suppressed A549 cell viability and activated apoptosis by NO level elevation. We concluded that AVCR NP plus DOX significantly induces A549 cytotoxicity-mediated apoptosis more than Huh-7 and HepG2 cells. This drug-drug nano-combination induced Huh-7 cytotoxicity-mediated apoptosis more than HepG2 cells. In conclusion, AVCR NP sensitized DOX-treated A549 and Huh-7 cells through reactive oxygen species (ROS)-stimulated apoptosis. Taken together, our data suggested that the CR plus AV nano-platforms would be a potential personalized medicine-based strategy for treating CCR2-positive NSCLC and HCC patients in the near future.

Keywords: Bevacizumab (avastin); CCR2 antagonist; Cytotoxicity; Hepatocellular carcinoma; non-small cell lung cancer.

Figure 1

The Cytotoxic and Apoptotic Effects of the AV and CR Therapeutic Regimens against HepG2 Cancer Cell Line. A, HepG2 cell viability (%) upon DOX, AV, DOX+AV, AVNP, and DOX+AVNP treatments (n=3); B, HepG2 cell viability (%) upon CR, AVCR, CRNP, AVCRNP, and DOX+AVCRNP treatments (n=3). The doses used for cytotoxicity were 0, 25, 50, 75, and 100 (µM for AV and DOX, and nM for CR); C, Representative images of HepG2 cell apoptosis (annexin versus PI) using flow cytometry upon DOX, AVNP, CRNP, and DOX+AVCRNP treatments; D, E, Mean and standard error (n=3) of HepG2 cancer cell early and late apoptosis, as well as necrosis versus control upon DOX, AVNP, CRNP, and DOX+AVCRNP treatments. The used dose for the flow cytometry experiments was either the IC₅₀ dose for the drugs which recorded IC₅₀ or 100 µM for DOX.The cells were incubated with the drugs for 24 h

Figure 2

The Cytotoxic and Apoptotic Effects of the AV and CR Therapeutic Regimens against Huh-7 Cancer Cell Line. A, Huh-7 cell viability (%) upon DOX, AV, DOX+AV, AVNP, and DOX+AVNP treatments (n=3); B, Huh-7 cell viability (%) upon CR, AVCR, CRNP, AVCRNP, and DOX+AVCRNP treatments (n=3). The doses used for cytotoxicity were 0, 25, 50, 75, and 100 (µM for AV and DOX, and nM for CR); C, Representative images of Huh-7 cell apoptosis (annexin versus PI) using flow cytometry upon DOX, AVNP, CRNP, and DOX+AVCRNP treatments; D, E, Mean and standard error (n=3) of Huh-7 cancer cell early and late apoptosis, as well as necrosis versus control upon DOX, AVNP, CRNP, and DOX+AVCRNP treatments. The used dose for the flow cytometry experiments was either the IC₅₀ dose for the drugs which recorded IC₅₀ or 100 µM for DOX.The cells were incubated with the drugs for 24 h

Figure 3

The Cytotoxic and Apoptotic Effects of the AV and CR Therapeutic Regimens against A549 Cancer Cell Line. A, A549 cell viability (%) upon DOX, AV, DOX+AV, AVNP, and DOX+AVNP treatments (n=3); B, A549 cell viability (%) upon CR, AVCR, CRNP, AVCRNP, and DOX+AVCRNP treatments (n=3). The doses used for cytotoxicity were 0, 25, 50, 75, and 100 (µM for AV and DOX, and nM for CR); C, Representative images of A549 cell apoptosis (annexin versus PI) using flow cytometry upon DOX, AVNP, CRNP, and DOX+AVCRNP treatments; D, E, Mean and standard error (n=3) of A549 cancer cell early and late apoptosis, as well as necrosis versus control upon DOX, AVNP, CRNP, and DOX+AVCRNP treatments. The used dose for the flow cytometry experiments was either the IC₅₀ dose for the drugs which recorded IC₅₀ or 100 µM for DOX.The cells were incubated with the drugs for 24 h

Figure 4

The Glucose Consumption Rate of A549 and Huh-7 Cancer Cells upon AV, CR, AVNP, CRNP, and DOX+AVCRNP. Mean and standard error (n=3) were represented in the blot. The used concentrations over cells were as follow: 0, 25, and 100 (µM for AV and DOX, and nM for CR). The cells were incubated with the drugs for 24 h

Figure 5

The Nitric Oxide (NO), Malondialdehyde (MDA), and Zinc (Zn) Levels of A549 and Huh-7 Cancer Cells upon AV, CR, AVNP, CRNP, and DOX+AVCRNP. Mean and standard error (n=3) were represented in the blot. The used concentrations over cells were as follow: 0, 25, and 100 (µM for AV and DOX, and nM for CR). The cells were incubated with the drugs for 24 h