Chandra Kirana

Antitumor activity of extract of Zingiber aromaticum and its bioactive sesquiterpenoid zerumbone

The anticancer properties of zerumbone (2,6,9 humulatriene-8-one, a sesquiterpenoid) from Zingiber aromaticum were compared with those of curcumin from Curcuma longa in an in vitro MTT tetrazolium salt assay using HT-29, CaCo-2, and MCF-7 cancer cells and in an azoxymethane (AOM)-induced animal model of colon cancer using aberrant crypt foci (ACFs) as a preneoplastic marker. The IC50 of zerumbone was approximately 10 μM and that of curcumin was 25 μM. Cell cycle arrest in HT-29 cells was observed at G0/G1 for 10 and 12.5 μMand G2/M for 25 μafter 24 h at concentrations of 10-25 μM of zerumbone, and a concentration-dependent increase in apoptosis (2-6% of viable cells) was observed after 48 h using the same concentration range. Male Sprague-Dawley rats were fed extracts in an AIN diet prepared from the equivalent of 4% by weight of dried rhizomes of Z. aromaticum and C. longa. ACFs were induced by two doses (15 mg/kg body weight) subcutaneously of AOM1 wk apart, the rats were killed 10 wk later, and the ACFs were assessed in the colon. Total ACFs were significantly reduced by Z. aromaticum extract (down 21%, P < 0.05) relative to control, the effect being most evident with large ACFs (≥3 aberrant crypts per focus). Similar reductions were observed with 4% C. longa extract in the diet (down 24%, P < 0.01) and with 2,000 ppm curcumin, the effect being particularly evident with large ACFs. The concentration of zerumbone in the Z. aromaticum extract diet was assayed at 300 ppm and of curcumin in the C. longa extract diet was also 300 ppm, i.e., the extract of C. longa was as effective at one-seventh the concentration of curcumin as the positive control. Zerumbone is effective as an anticancer agent, possibly by its apoptosis-inducing and antiproliferative influences. This latter possibility is currently being investigated.

Screening for Antitumor Activity of 11 Species of Indonesian Zingiberaceae Using Human MCF-7 and HT-29 Cancer Cells

We have screened 11 important species of Zingiberaceae, used as spices and for medicinal purposes in Indonesia, for their antitumor activity using human HT-29 colon cancer and MCF-7 breast cancer cells. They were Amommum cardamomum, Curcuma aeruginosa, C. longa, C. mangga, C. xanthorrhiza, Kaempferia galanga, K. pandurata, K. rotunda, Z. aromaticum, Z. cassumunar, and Zingiber officinale. Ethanol extracts of eight species showed strong inhibitory effect on the growth of the cancer cells when evaluated using the colorimetric tetrazolium salt assay. Since curcumin, a yellow pigment isolated from C. longa, has shown its potential anticancer activity in vitro and in vivo studies and is currently undergone clinical trial in the US, we used an extract of C. longa as a comparison. Extracts of K. pandurata and Z. aromaticum had very strong inhibitory activity against the two cell lines similar to those of C. longa. However, curcumin was not detectable in the extracts of those two plants. The ethanol extracts of the active species had less effect on the growth of a non-transformed human skin fibroblast cell line (SF 3169). Microscopic examination of cancer cells exposed to extracts of active species showed a characteristic morphology of apoptosis. Further study on Z. aromaticum and K. pandurata, including identification of bioactive compounds and elucidation of mechanism(s) likely to be operating, has been carried out.

Cathepsin D Expression in Colorectal Cancer: From Proteomic Discovery through Validation Using Western Blotting, Immunohistochemistry, and Tissue Microarrays.

Despite recent advances in surgical techniques and therapeutic treatments, survival from colorectal cancer (CRC) remains disappointing with some 40–50% of newly diagnosed patients ultimately dying of metastatic disease. Current staging by light microscopy alone is not sufficiently predictive of prognosis and would benefit from additional support from biomarkers in order to stratify patients appropriately for adjuvant therapy. We have identified that cathepsin D expression was significantly greater in cells from invasive front (IF) area and liver metastasis (LM) than those from main tumour body (MTB). Cathepsin D expression was subsequently examined by immunohistochemistry in tissue microarrays from 119 patients with CRC. Strong expression in tumour cells at the IF did not correlate significantly with any clinico-pathological parameters examined or patient survival. However, cathepsin D expression in cells from the MTB was highly elevated in late stage CRC and showed significant correlation with subsequent distant metastasis and shorter cancer-specific survival. We also found that macrophages surrounding tumour cells stained strongly for cathepsin D but there was no significant correlation found between cathepsin D in macrophages at IF and MTB of CRC patient with the clinic-pathological parameters examined.

Compatibility of toluidine blue with laser microdissection and saturation labeling DIGE

Tissue fixation and staining protocols for laser microdissection are frequently not fully compatible with subsequent proteomic analysis. We compared the effect of three common histological stains (toluidine blue (TB), hemotoxylin, and hematoxylin and eosin (HE)) on tissue visualization, protein recovery, the saturation labeling reaction, and 2‐D electrophoresis. TB provided the best visualization of colorectal tumor tissue during laser microdissection (LMD) and had a comparable effect on protein recovery and the saturation labeling reaction with hematoxylin, provided a modified 2‐D clean‐up protocol was used. Eosin inhibited both protein recovery and the saturation labeling reaction.

TUFM is a potential new prognostic indicator for colorectal carcinoma

Aims: Mitochondrial Tu translation elongation factor (TUFM) is a nuclear encoded protein that participates in mitochondrial polypeptide translation. TUFM has been reported to be over-expressed in many tumour types including colorectal carcinoma (CRC) by proteomics. The present study aims to examine the prognostic implication of TUFM in CRC. Methods: Immunohistochemical staining was performed in tissue microarrays composed of 123 cases of CRC using a polyclonal anti-TUFM antibody. Immunoreactivity was quantified using Image-Pro plus software, and analysed in association with patients’ clinicopathological parameters and survival time. Results: The immunoreactivity of TUFM was negative in 25%, weak in 50% and strong in 25% of CRC cases. TUFM immunoreactivity had no significant association with the clinicopathological parameters examined including TNM stage and grade. However, strong TUFM expression significantly correlated with a higher 5-year recurrence rate (p = 0.024). Kaplan–Meier analysis revealed that patients with strong TUFM expression had significantly shorter cancer-specific survival than patients with negative TUFM (log-rank test, p = 0.038). In multivariate analysis, strong TUFM expression remained a stage-independent unfavourable prognostic indicator (p = 0.024). Conclusions: Increased expression of TUFM is a promising new prognostic indicator for CRC. Selective inhibition of TUFM in tumour cells may present a new avenue for the targeted therapy of this cancer.

Soluble HLA-G is a differential prognostic marker in sequential colorectal cancer disease stages: sHLA-G in the Differential Prognosis of CRC

The expression of HLA‐G by tumour cells is an established mechanism to escape recognition and immune mediated destruction, allowing tumour survival, growth and metastasis. However, the prognostic value of soluble HLA‐G (sHLA‐G) remains unknown. Mucinous carcinoma (MC) is a distinct form of colorectal cancer (CRC) found in 10 to 15% of patients, which has long been associated with poor response to treatment. To investigate the prognostic value of plasma sHLA‐G levels in CRC patients, preoperative plasma sHLA‐G levels were determined by ELISA in CRC patients (n = 133). In addition, the local expression of HLA‐G in tumour biopsies was assessed using tissue microarray analysis (n = 255). Within the high 33rd percentile of sHLA‐G levels (265–890 U/mL; n = 44) we observed higher frequency of MC patients (p = 0.012; Chi‐square), and higher sHLA‐G levels in patients with vascular invasion (p = 0.035; two‐tailed t‐test). Moreover, MC patients had significantly higher sHLA‐G levels compared to those with adenocarcinoma not otherwise specified (p = 0.036; two‐tailed t‐test). Surprisingly, while stage II patients showed negative correlation between sHLA‐G levels and liver metastasis free survival (LMFS) (p = 0.041; R = −0.321), in stage III patients high sHLA‐G levels were associated with significantly longer LMFS (p = 0.002), and sHLA‐G levels displayed positive correlation with LMFS (p = 0.006; R = 0.409). High HLA‐G expression in tumours was associated with poor cancer specific overall survival in stage II to III (p = 0.01), and with shorter LMFS in stage II patients (p = 0.004). Our findings reveal that sHLA‐G levels are associated with distinct progression patterns in consecutive disease stages, indicating a potential value as surrogate marker in the differential prognosis of CRC.

Discordant frequencies of tissue-resident and circulating CD180-negative B cells in chronic rhinosinusitis

The unconventional toll‐like receptor (TLR) CD180 is implicated in chronic inflammatory diseases; however, its role in chronic rhinosinusitis (CRS) has yet to be investigated. Here we study the expression of CD180, its homologue TLR4 and myeloid differentiation factor 1 (MD1) on mucosal and systemic immune cell populations in relation to serum immunoglobulin G (IgG) levels.