Antitumor activity of noscapine in human non-small cell lung cancer xenograft model

Abstract

Purpose: An antitussive plant alkaloid, Noscapine HCl (Nos) displays anticancer activity and has a safe pharmacological profile in humans. The current study was aimed to investigate the in vitro and in vivo anti tumor activity of Nos to determine possible mechanisms of anti tumor activity for treatment of non-small cell lung cancer (NSCLC).

Methods: In vitro cytotoxicity of Nos was studied in H460 cells treated with different doses of Nos (10-160 microM) for 72 h and cell viability was determined using crystal violet assay. Apoptosis in H460 cells was evaluated by TUNEL assay after treatment of cells for 72 h with 30 and 40 microM doses of Nos. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and on day 4 onwards Nos was administered orally at dose of 300, 450 and 550 mg/kg/day for 24 days. As a control, xenografted tumors were separately treated with Docetaxel (10 mg/kg i.v. bolus on day 5, 11, 17, 23). The tumor volumes were measured every five days. Expression of PARP, Bcl(2, )Bax, and caspase-3 families of proteins was measured by Western Blotting (WB), while TUNEL and Immunohistochemical methods were utilized to determine DNA fragmentation and cleaved caspase-3 levels respectively.

Results: Nos inhibited growth of H460 cells with the IC50 values of 34.7 +/- 2.5 microM. Nos at 30 and 40 microM doses caused apoptosis as evidenced by nuclear condensation in treated H460 cells. Nos caused 49, 65 and 86% reduction in the xenografted tumor volumes at a dose of 300 (P < 0.05), 450 (P < 0.01), 550 mg/kg/day (P < 0.01), respectively, when compared to controls. Nos-dependent suppression of xenografted tumor growth involved up regulation of PARP, Bax, caspase-3 and repression of Bcl(2) expression. An increase in Bax/Bcl(2) ratio suggests involvement of a mitochondrial mediated apoptotic processes. Our studies revealed a non significant (P > 0.05) increase in Bax/Bcl(2) ratio with Nos at a dose of 300 mg/kg/day, while a significant (P < 0.001) increase in Bax/Bcl(2) ratio was observed with Nos doses of 450 and 550 mg/kg/day. Further, Nos caused elevated apoptosis in tumor xenografts as evidenced by enhanced expression of caspase-3 and positive TUNEL staining in regressed tumor tissues, thus suggesting induction of apoptosis by mitochondrial pathway.

Conclusion: Our studies suggest that potent antitumor activity of Nos against NSCLC cells. Oral administration of Nos showed significant reduction in tumor volume in human non-small cell lung tumor xenograft in nude mice in a dose dependant manner. Thus, Nos is a promising novel chemotherapeutic agent for the treatment of human lung cancer.

Fig. 1

In vitro cytotoxicity profiles of Nos in H460 cells. Cells were treated with Nos and cytotoxicity was determined using the crystal violet assay as described in materials and methods. A plot of the percentage of cell survival versus Nos concentrations used for the determination of IC 50 values. Results are expressed as means ± SD for at least three separate determinations

Fig. 2

Fluorescence micrographs of H460 cells stained with rhodamine and DAPI for control cells (A1, A2), 72-h treatment with Nos 30 μM (B1, B2), and Nos 40 μM (C1, C2). DNA fragmentation indicated by positive staining and nuclear condensation indicated by DAPI nuclear staining. Micron bar 10 μm

Fig. 3

In vivo antitumor effect of different doses of Nos compared to untreated and Docetaxel treated (positive control) in H460 lung tumor Xenograft model. (a) progression profile of tumor growth kinetics and (b) tumor volume measurements on days 28 post-inoculation. (tumor volumes, mm³ ± SEM)

Fig. 4

Measurement of body weight following different doses of Nos compared to untreated and Docetaxel treated (positive control) mice

Fig. 5

Western blotting of tumor tissue lysates to determine expressions apoptosis-related proteins. (a). The tumors were harvested 28 days post tumor implantation, lysates were prepared, and a 50 μg of protein was loaded in each lane. Lane 1 control, Lane 2 Docetaxel 10 mg/kg i.v. bolus, Lane 3 Nos 300 mg/kg/day, Lane 4 Nos 450 mg/kg/day and Lane 5 Nos 550 mg/kg/day. The densitometric analysis of the bands to determine level of expression of PARP (b), Caspase-3 (c), Bax (d), and Bcl₂ (e) in xenograft human H40 lung tumors. The densitometric analysis of the bands was performed using the program ImageJ v1.33u. Error bars depict standard deviations ***P < 0.001 versus untreated control

Fig. 6

Nos inhibits tumor growth by triggering apoptosis. a–c, Activated (cleaved) caspase-3 was examined by immunohistochemical staining of paraffin-embedded tumor sections, from untreated control (a) and Nos-treated (b and c) mice. d–f Representative TUNEL stained micrographs of tumor sections from untreated control (d) and Nos-treated (e, f) mice