PDF - 89K, HPLC and Mass spectrometric analysis of mice plasma and tumor tissues for the bioavailability of SENL compounds after oral administration of SENL, 200 mg/kg body weight. A, HPLC chromatogram of plasma of mice treated with SENL. B, HPLC chromatogram of LNCaP xenograft tumor tissue of mice treated with SENL. C, The mass spectrometric analysis of mice tumor tissue fraction 1 obtained from HPLC depicts the M ion at 488.1824 m/z, suggestive by mass alone as the sodium salt of nimbolide. D, The mass spectrometric analysis of mice tumor tissue fraction 2 obtained from HPLC depicts the M ion at 452.2195 m/z, suggestive by mass alone as the 28-deoxonimbolide. HPLC and mass spectrometric analysis was performed in duplicate, representative chromatograms were shown. The difference between the measured value for the sodium salt of nimbolide standard from the measured value in plasma and tumor tissue is less than 2.8 ppm and for 28-deoxonimbolide in the tumor tissues from the calculated value is less than 1 ppm.
ARTICLE ABSTRACT
Azadirachta indica, commonly known as neem, has gained worldwide prominence because of its medical properties, namely antitumor, antiviral, anti-inflammatory, antihyperglycemic, antifungal, and antibacterial activities. Despite these promising results, gaps remain in our understanding of the molecular mechanism of action of neem compounds and their potential for use in clinical trials. We investigated supercritical extract of neem leaves (SENL) for the following: molecular targets in vitro, in vivo efficacy to inhibit tumor growth, and bioactive compounds that exert antitumor activity. Treatment of LNCaP-luc2 prostate cancer cells with SENL suppressed dihydrotestosterone-induced androgen receptor and prostate-specific antigen levels. SENL inhibited integrin β1, calreticulin, and focal adhesion kinase activation in LNCaP-luc2 and PC3 prostate cancer cells. Oral administration of SENL significantly reduced LNCaP-luc2 xenograft tumor growth in mice with the formation of hyalinized fibrous tumor tissue, reduction in the prostate-specific antigen, and increase in AKR1C2 levels. To identify the active anticancer compounds, we fractionated SENL by high-pressure liquid chromatography and evaluated 16 peaks for cytotoxic activity. Four of the 16 peaks exhibited significant cytotoxic activity against prostate cancer cells. Mass spectrometry of the isolated peaks suggested the compounds with cytotoxic activity were nimbandiol, nimbolide, 2′,3′-dihydronimbolide, and 28-deoxonimbolide. Analysis of tumor tissue and plasma samples from mice treated with SENL indicated 28-deoxonimbolide and nimbolide as the bioactive compounds. Overall, our data revealed the bioactive compounds in SENL and suggested that the anticancer activity could be mediated through alteration in androgen receptor and calreticulin levels in prostate cancer. Mol Cancer Ther; 13(5); 1067–77. ©2014 AACR.
