ANTI PROLIFERATIVE ACTIVITY OF CALAMUS ROTANG AS A SPOTLIGHT ON EHRLICH'S ASCITES CARCINOMA TREATED PERITONEAL AS WELL AS SOLID TUMOR MODEL

Objective: Methanol extract of Calamus rotang (MECR) root was appraised as a spotlight for the candidate of anticancer activity through the vehicle (Ehrlich Ascites Carcinoma) on Swiss albino mice.Methods: In vitro cytotoxicity assay has been accessed by trypan blue and MTT assay. In vivo anticancer activity was done using EAC cells (2 × 106) where in each groups mice were 6. After treatment with MECR at the lower dose of 200 and higher dose of 400 mg/kg respectively for 9 d, half of the mice of each group were sacrificed and the rest were kept to check prolongation of life span. The anticancer potential of MECR was evaluated by tumor volume, viable and nonviable tumor cell count, tumor weight, hematological parameters, biochemical estimations and Furthermore, tissue antioxidant parameters. Besides, solid tumor activity was also inspected.Results: In MECR treated groups (200 and 400 mg/kg) tumor volume, packed cell volume and viable cell count was significantly lessened as compared to that of the EAC control group. Life span, most reliable criteria for anticancer study, increased quite surprisingly by 50% and 100% in a dose dependant manner while compared to EAC control group. The hematological, biochemical and liver tissue antioxidant parameter are significantly (p<0.05) restored along with solid tumor case study (solid tumor volume) towards the normal level after treatment with MECR.Conclusion: From the above study it can be inferred that the MECR has impressive anticancer activity in dose dependent way

Donor Hematopoietic Stem Cells Confer Long-Term Marrow Reconstitution by Self-Renewal Divisions Exceeding to That of Host Cells

<div><p>Dormant hematopoietic stem cells (HSCs) are activated by microenvironmental cues of the niche in response to the injury of bone marrow (BM). It is not clearly understood how engrafted cells respond to these cues and are involved in marrow regeneration. The purpose of this study was to decipher this cellular response in competitive environment. BM cells of CD45.2 mice were transplanted in sub-lethally irradiated CD45.1 mice. The status of the donor and recipient stem cells (LSK: Lin⁻Sca-1⁺c-Kit⁺) were determined by flowcytometry using CD45 alleles specific antibodies. The presence of long-term engraftable stem cells was confirmed by marrow repopulation assay in secondary hosts, and cell cycle status was determined by staining with Ho33342 and pyronin Y, and BrdU retention assay. The expressions of different hematopoietic growth factor genes in stromal compartment (CD45⁻ cells) were assessed by real-time reverse transcriptase- polymerase chain reaction (RT-PCR). The presence of donor cells initially stimulated the proliferation of host LSK cells compared with control mice without transplantation. This was expected due to pro-mitotic and anti-apoptotic factors secreted by the donor hematopoietic cells. Upon transplantation, a majority of the donor LSK cells entered into cell cycle, and later they maintained cell cycle status similar to that in the normal mouse. Donor-derived LSK cells showed 1000-fold expansion within 15 days of transplantation. Donor-derived cells not only regenerated BM in the primary irradiated host for long-term, they were also found to be significantly involved in marrow regeneration after the second cycle of irradiation. The proliferation of LSK cells was associated with the onset of colossal expression of different hematopoietic growth factor genes in non-hematopoietic cellular compartment. Activation of donor LSK cells was found to be dynamically controlled by BM cellularity. Long-term study showed that a high level of hematopoietic reconstitution could be possible by donor cells in a sub-lethally irradiated host.</p> </div

BM donor cells chimerism in irradiated mice.

<p>Experiment was initiated as before, after 7 months of transplantation mice were subjected to second cycle of irradiation and maintained for 1 month. Mice were sacrificed and BM cells were analyzed for chimerism. Representative dot-plot show 78% donor (CD45.2) cells chimerism (n = 3).</p

Proliferation of LSK cells during marrow regeneration.

<p>(A) Sub-lethally irradiated mice were maintained (without transplantation) for different time points. Mice were sacrificed and LSK cells were determined by flowcytometry. Bar diagram shows proliferation kinetics of host BM-LSK cells (n = 6, each time point); (B) Each sub-lethally irradiated mouse received 10×10⁶ crude BM cells of CD45.2 mouse. Mice were sacrificed and LSK cells were determined by flowcytometry. Bar diagram shows proliferation kinetics of host and donor LSK cells (n = 6, each time point); (C) Each sub-lethally irradiated mouse received 3×10⁴ CD45.2LSK cells. Mice were sacrificed and LSK cells were determined by flowcytometry. Bar diagram shows proliferation kinetics of host and donor LSK cells (n = 3, each time point); (D, E) Competitive marrow repopulation assay. Donor hematopoietic (CD45.2⁺) cells of 10 and 15 days of transplantation were isolated from primary recipient. Four different doses of above sorted cells (10×10³, 30×10³, 100×10³ and 300×10³) were transplanted in 4 groups of mice, each consisting of 4. After 1 month of transplantation, BM cells were isolated from secondary recipient and analyzed for donor-derived LSK cells. Proportional increase in the recovery of donor LSK with transplantation dose are shown in E.</p

Effect of normal hematopoietic cells on proliferation and anti-apoptotic effects in irradiated bone marrow cells in culture (direct or indirect contact).

<p>Above values were calculated from flowcytometric analyses of respective samples (n = 2 to 3). Representative dot-plots of flowcytometric analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050693#pone.0050693.s005" target="_blank">Figures S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050693#pone.0050693.s006" target="_blank">S6</a>.</p

Anti-apoptotic effect of normal donor cells on irradiated host cells.

<p>Number of mice in each time point was 3 to 4.</p

Pulse-chase experiment for nuclear incorporation of BrdU.

<p>After transplantation as above, a group of mice were given BrdU pulse for 10 days (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050693#s2" target="_blank">Materials and Methods</a>), which was followed by chase for 20 days. First LSK cells were sorted and then stained for BrdU. The representative histogram (top) show 70.5% of donor (D) and 17.1% of recipient (R) LSK cells were labelled with BrdU. The representative histogram (bottom) shows 98% of recipient and 89% of donor cells retained BrdU. C: Control LSK cells (n = 3).</p

Donor-derived hematopoietic progenitor cells are localized in the trabicular bone.

<p>Sub-lethally irradiated mice were transplanted with GFP-expressing crude BM cells. After a month, mice were sacrificed; longitudinal trabicular bone cryosections (5 µm) were obtained. Sections were stained for osteopontin (AF594, red), GFP (AF488, green), Sca-1 (AF555, pink) and nuclei (DAPI, blue). Both immunofluorescence (composite) and bright filed confocal images are shown. Arrows indicating donor derived progenitor cells.</p

Expression of cyclin A and <i>p21</i>^cip1/waf1 in CD45.1LSK cells.

<p>(A) Sub-lethally irradiated CD45.1 mice were transplanted with or without CD45.2LSK Cells. Mice were sacrificed after 3 days of transplantation and host LSK cells were sorted and immuno- stained for cyclin A protein. Representative images show that a few cells expressed cyclin A (arrow) in untransplanted mouse than transplanted (magnification 60×); (B) Host LSK cells of above two groups were compared for the expression of <i>p21</i> gene by real time RT-PCR. Bar diagram shows relative ΔCt values in two different conditions (n = 3).</p

Kinetics of gene expression by real time RT-PCR.

<p>BM cells were harvested at different time points following irradiation and transplantation; stromal cells (CD45⁻) were sorted and analyzed for relative (fold) expression of the hematopoietic growth factor genes compared to that in unirradiated and untransplanted mice. Bar diagrams show fold expressions of mRNAs for different growth factors at days 1, 2, 3, 5 and 15 of transplantation (n = 3).</p