Excised Tumor Tissue Research Articles

Congenital Dermatofibrosarcoma Protuberans in a Newborn Infant With a Massive Back Tumor

Dermatofibrosarcoma protuberans (DFSP) is known as a very rare malignant tumor of the deep dermis and subcutaneous tissue. It typically develops during adolescence and adulthood, with pediatric and infantile cases, particularly congenital ones, being much less frequent. We report a neonate with congenital DFSP. A newborn girl presented with a massive back tumor at birth. The tumor was at first suspected to be infantile fibrosarcoma (IFS) after immunohistochemical analysis of biopsy material, although the results were not fully compatible with IFS. She received chemotherapy under a tentative diagnosis of IFS, but this was unsuccessful. Partial resection was therefore performed at the age of 8 months to reduce the tumor mass and to reexamine its immunohistochemical characteristics. Positive CD34 staining and Collagen α1α/platelet-derived growth factor beta chimera gene signals on analysis of the excised tumor tissues enabled a definitive diagnosis of DFSP. She then underwent local irradiation and was given a daily dose of oral tyrosine kinase inhibitor (imatinib). After almost 1 year, the patient is doing well without enlargement of the residual tumor.

An ErbB-3 antibody, MP-RM-1, inhibits tumor growth by blocking ligand-dependent and independent activation of ErbB-3/Akt signaling

The ErbB receptors, such as ErbB-1 and ErbB-2, have been intensely pursued as targets for cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance, which is often associated with reactivation of the ErbB-3-PI3K-Akt signaling. This may be overcome by an ErbB-3 ligand that abrogates receptor-mediated signaling. Toward this end, we have generated a mouse monoclonal antibody, MP-RM-1, against the extracellular domain (ECD) of ErbB-3 receptor. Assessment of human tumor cell lines, as well as early passage tumor cells revealed that MP-RM-1 effectively inhibited both NRG-1β-dependent and -independent ErbB-3 activation. The antagonizing effect of MP-RM-1 was of non-competitive type, as binding of [(125)I]-labeled NRG-1β to ErbB-3 was not influenced by the antibody. MP-RM-1 treatment led, in most instances, to decreased ErbB-3 expression. In addition, MP-RM-1 was able to inhibit the colony formation ability of tumor cells and tumor growth in two human tumor xenograft nude mouse models. Treatment with the antibody was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Collectively, these results indicate that MP-RM-1 has the potential to interfere with signaling by ErbB-3 and reinforce the notion that ErbB-3 could be a key target in cancer-drug design.

In vivo three-dimensional spectroscopic photoacoustic imaging for monitoring nanoparticle delivery.

In vivo monitoring of nanoparticle delivery is essential to better understand cellular and molecular interactions of nanoparticles with tissue and to better plan nanoparticle-mediated therapies. We developed a three-dimensional ultrasound and photoacoustic (PA) imaging system and a spectroscopic PA imaging algorithm to identify and quantify the presence of nanoparticles and other tissue constituents. Using the developed system and approach, three-dimensional in vivo imaging of a mouse with tumor was performed before and after intravenous injection of gold nanorods. The developed spectroscopic PA imaging algorithm estimated distribution of nanoparticle as well as oxygen saturation of blood. Moreover, silver staining of excised tumor tissue confirmed nanoparticle deposition, and showed good correlation with spectroscopic PA images. The results of our study suggest that three-dimensional ultrasound-guided spectroscopic PA imaging can monitor nanoparticle delivery in vivo.

Establishment of a Primary Pleomorphic Xanthoastrocytoma Cell Line

Anaplastic pleomorphic xanthoastrocytoma is an aggressively growing, malignant, and eventually fatal tumor of the central nervous system. Testing chemotherapeutic drug sensitivity under in vitro conditions would be a useful strategy to determine sensitive or resistant drugs for fatal brain cancers. To establish primary cell cultures of excised tumor tissue from pleomorphic xanthoastrocytoma-bearing patients and to test their sensitivity against various anticancer chemotherapy drugs. Prepared suspensions of the excised tumor tissue from a patient who had a recurrent grade 3 pleomorphic xanthoastrocytoma was cultured in culture dishes until cells began to grow. Immunofluorescent and immunohistochemical visualizations were performed using confocal and light microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in comparison with ³H-thymidine incorporation assay was used to test cellular toxicity of several anticancer drugs. We established vigorously growing primary cells of the tumor. Drug sensitivity testing was conducted successfully. Primary cell cultures of surgically removed tumor tissues may be useful in studies of cancer biology and chemotherapeutic drug sensitivity for recurrent malignant brain tumors, particularly for anaplastic pleomorphic xanthoastrocytoma.

Discriminant analysis to classify glioma grading using dynamic contrast-enhanced MRI and immunohistochemical markers

The purpose of the present study was to look for the possible predictors which might discriminate between high- and low-grade gliomas by pooling dynamic contrast-enhanced (DCE)-perfusion derived indices and immunohistochemical markers. DCE-MRI was performed in 76 patients with different grades of gliomas. Perfusion indices, i.e., relative cerebral blood volume (rCBV), relative cerebral blood flow (rCBF), permeability (k (trans) and k (ep)), and leakage (v (e)) were quantified. MMP-9-, PRL-3-, HIF-1α-, and VEGF-expressing cells were quantified from the excised tumor tissues. Discriminant function analysis using these markers was used to identify discriminatory variables using a stepwise procedure. To look for correlations between immunohistochemical parameters and DCE metrics, Pearson's correlation coefficient was also used. A discriminant function for differentiating between high- and low-grade tumors was constructed using DCE-MRI-derived rCBV, k (ep), and v (e). The form of the functions estimated are "D (1) = 0.642 × rCBV + 0.591 × k (ep) - 1.501 × v (e) - 1.550" and "D (2) = 1.608 × rCBV + 3.033 × k (ep) + 5.508 × v (e) - 8.784" for low- and high-grade tumors, respectively. This function classified overall 92.1% of the cases correctly (89.1% high-grade tumors and 100% low-grade tumors). In addition, VEGF expression correlated with rCBV and rCBF, whereas MMP-9 expression correlated with k (ep). A significant positive correlation of HIF-1α with rCBV and VEGF expression was also found. DCE-MRI may be used to differentiate between high-grade and low-grade brain tumors non-invasively, which may be helpful in appropriate treatment planning and management of these patients. The correlation of its indices with immunohistochemical markers suggests that this imaging technique is useful in tissue characterization of gliomas.

Differential Expression of the 18 kDa Translocator Protein (TSPO) by Neoplastic and Inflammatory Cells in Mouse Tumors of Breast Cancer

Tumor-associated inflammation has been linked to angiogenesis, metastasis and poor prognosis. The 18 kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor (PBR), is expressed in activated immune cells such as macrophages, but also in a number of cancer cell lines such as those of breast cancer. There is an increasing clinical interest in TSPO expression as it has been proposed as a poor prognostic factor for survival in lymph-node negative breast cancer patients. This study aims to assess of the presence of neoplastic cell-associated TSPO and tumor macrophage-associated TSPO in mouse xenografts generated from the MDA-MB-231 and the MCF-7 breast cancer cell lines, as well as 25 different breast tumors originally derived from patient-tissue but propagated in mice using two antibodies, each specific to either the human or the murine form of TSPO. Autoradiography with the TSPO ligand [¹⁸F]DPA-714 and immunohistochemistry were also performed on the excised tumor tissues from the MDA-MB-231, MCF-7 and one of the patient-derived xenografts (HBCx-12B). High TSPO expression (either cancer or stromal cell-associated, or both) was measured in 20/25 (80%) of the patient-derived breast cancer xenografts. [¹⁸F]DPA-714 showed displaceable binding to both the human and murine TSPO on tumor tissue sections. Immunohistochemistry demonstrated that a significant portion of the tumor stromal TSPO expression colocalized with F4/80 positive macrophages cells. This study constitutes a first report of the tumor TSPO expression by mixed cell populations, and it may have important implications for cancer biology as well as for the development of imaging and therapeutic ligands targeted to TSPO.

Abstract PD02-06: New Molecular Biomarkers for Resistance to Trastuzumab in Primary HER2 Positive Breast Cancer — A Translational Investigation from the Neoadjuvant GeparQuattro Study

Abstract Background: Little is known about the mechanisms of resistance to trastuzumab (T). We investigated markers which may indicate resistance to trastuzumab in a cohort of HER2 positive patients treated in a neoadjuvant phase 3 trial. Patients and methods: In the neoadjuvant GeparQuattro trial 1495 patients with operable and locally advanced tumors were randomized to 3 different chemotherapy regimens (4 x EC ≥4 x docetaxel (D) vs 4 x EC ≥4 x D/capecitabine (C) vs 4 x EC ≥4x D ≥4 X C). All HER2 positive patients (n=445 assessed by local testing) received trastuzumab concurrently with chemotherapy. 216 pretherapeutic core biopsies were available for tissue microarrays (TMA). For this evaluation only cases with sufficient tumor tissue on TMA (>30%) and positive HER2 status by central reassessment (IHC and CISH) were used (n=153). Expression of different markers possibly associated with resistance to T-based treatment were immunohistochemically (IHC) investigated. Marker expression was assessed with Immunoreactive-Score (IRS) combining intensity and % of positive cells. Resistance to treatment was defined as minimal or no microscopic changes in surgically excised tumor tissue (regression grade RG 0-1 according to Sinn et al). Results: The 153 centrally HER2 positive cases were taken for analysis. Median age was 49 years (22-78). Clinical T stadium was T1,2,3,4a-c/4d in 3%/70%/11%/8%/8% respectively, 59%/41% of these tumors were G2/G3; 51% hormone receptor (HR) positive. 44 (29%) did not show any response to treatment (RG 0-1) indicating resistance. In the chi2 test (2-sided) the following parameters were significantly associated with resistance (RG0- 1) to combined trastuzumab-cytotoxic therapy: insulin like growth factor receptor-1 (IGFR-1; IRS >3 resistant; p=0.029, insulin receptor (IR: IRS >1 resistant; p=0.028), Notch1(IRS>8 resistant; p= 0.037), ALDH1(IRS > 4 resistant; p=0.001), p4E-BP1 (IRS> 4resistant; p =0.017), p27 (IRS > 4 resistant; p=0.032). In multivariable testing (backward selection) ALDH 1 (OR 6.65 95%CI 1.78-24.76; p= 0.005) and p4E-BP1 (OR 3.2595% CI 1.36-9.06 p=0.009) remained significantly associated with resistance to trastuzumab containing therapy. Univariate and multivariate analysis for resistance markers to trastuzumab Conclusions: Our results suggest that an activated mTOR pathway characterized by increased levels of p4EBP1 and stem cell features described by enhanced ALDH1 expression are involved in resistance to trastuzumab containing treatment in primary HER2 positive breast cancer. These resistance markers will be validated in the neoadjuvant GeparQuinto trial investigating in a randomized design the effect of trastuzumab compared to lapatinib in HER2 positive primary breast cancer patients in addition to EC-D. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-06.

Assessment of early docetaxel response in an experimental model of human breast cancer using DCE-MRI, ex vivo HR MAS, and in vivo 1 H MRS

The purpose of this study was to evaluate the use of dynamic contrast-enhanced (DCE) MRI, in vivo (1)H MRS and ex vivo high resolution magic angle spinning (HR MAS) MRS of tissue samples as methods to detect early treatment effects of docetaxel in a breast cancer xenograft model (MCF-7) in mice. MCF-7 cells were implanted subcutaneously in athymic mice and treated with docetaxel (20, 30, and 40 mg/kg) or saline six weeks later. DCE-MRI and in vivo (1)H MRS were performed on a 7 T MR system three days after treatment. The dynamic images were used as input for a two-compartment model, yielding the vascular parameters K(trans) and v(e). HR MAS MRS, histology, and immunohistochemical staining for proliferation (Ki-67), apoptosis (M30 cytodeath), and vascular/endothelial cells (CD31) were performed on excised tumor tissue. Both in vivo spectra and HR MAS spectra were used as input for multivariate analysis (principal component analysis (PCA) and partial least squares regression analysis (PLS)) to compare controls to treated tumors. Tumor growth was suppressed in docetaxel-treated mice compared to the controls. The anti-tumor effect led to an increase in K(trans) and v(e) values in all the treated groups. Furthermore, in vivo MRS and HR MAS MRS revealed a significant decrease in choline metabolite levels for the treated groups, in accordance with reduced proliferative index as seen on Ki-67 stained sections. In this study DCE-MRI, in vivo MRS and ex vivo HR MAS MRS have been used to demonstrate that docetaxel treatment of a human breast cancer xenograft model results in changes in the vascular dynamics and metabolic profile of the tumors. This indicates that these MR methods could be used to monitor intra-tumoral treatment effects.

Application of Radiofrequency Ablation of Renal VX2 Tumors by Cooled-Tip Electrode in a Rabbit Model

This study has two purposes: (1) To compare the use of tumor cell suspensions and excised tumor tissue for generating renal tumors suitable for radiofrequency ablation (RFA) studies and (2) to assess the effectiveness of RFA for ablation of renal VX2 tumors in a rabbit model. VX2 tumor masses were used to inoculate 17 rabbits. The renal VX2 tumors of 13 rabbits were subsequently treated with a cooled-tip radiofrequency system at a power of 15 to 30 W for 3 to 6 minutes, and 4 rabbits served as controls. After RFA, rabbits were observed by contrast-enhanced CT to ascertain the ablative range. Rabbits were divided into three groups and sacrificed at 3, 7, and 14 days post-treatment for histopathologic studies. Both tumor implant methods had a 100% success rate in generating tumors. The average lesion produced was 1.3 x 1.8 x 1.5 cm. Complete tumor ablation occurred in 76.9%, and local tumor growth in 23.1%. Depending on the position of the tumors, the complications after RFA treatment included infarction, perirenal hematoma, hydronephrosis, and abscess. The overall complication rate was 38.5%. In histopathologic studies, a central needle track, tumor coagulation, renal tissue coagulation, peripheral hemorrhage, and an inflammatory layer could be observed. Application of RFA to destroy renal VX2 tumors in this rabbit model has a relatively high cure rate. It can be used to destroy small renal tumors in a precise and noninvasive manner. We hope to apply this procedure in selected human renal tumors in the future.

Application of Radiofrequency Ablation of Renal VX2Tumors by Cooled-Tip Electrode in a Rabbit Model.

Objective. This study has two purposes: (a) to compare the use of tumor cell suspensions and excised tumor tissue for generating renal tumors suitable for radiofrequency ablation (RFA) studies and (b) to assess the efficacy of RFA for ablation of renal VX2 tumors in a rabbit model. Procedures. VX2 tumor masses were used to inoculate 17 rabbits. The renal VX2 tumors of 13 rabbits were subsequently treated with a cooled-tip RF system at a power of 15-30 W for 3-6 minutes, and 4 rabbits served as control. After RFA, rabbits were observed by contrast-enhanced CT to ascertain the ablative range. Rabbits were divided into three groups and sacrificed at 3, 7, and 14 days post-treatment for histopathological studies. Results. Both tumor implant methods had a 100% success rate in generating tumors. The average lesion produced was 1.3x1.8x1.5 cm. Complete tumor ablation occurred in 76.9%, and local tumor growth in 23.1%. Depending on the position of the tumors, the complications after RFA treatment included infarction, perirenal hematoma, hydronephrosis, and abscess. The over-all complication rate was 38.5%. In histopathological studies, a central needle track, tumor coagulation, renal tissue coagulation, peripheral hemorrhage, and an inflammatory layer could be observed. Conclusion and clinical relevance. Application of RFA to destroy renal VX2 tumors in this rabbit model has a relatively high cure rate. It can be used to destroy small renal tumors in a precise and non-invasive manner. Impact on human medicine. We hope to apply this procedure in selected human renal tumors in the future.

VIPoma with expression of both VIP and VPAC1 receptors in a patient with WDHA syndrome

We report a case of VIPoma in a 72-year-old female patient who presented with excessive diarrhea, severe hypokalemia, and acidemia. She had been referred to our hospital three times because of severe diarrhea. No primary tumor site was found by conventional techniques, including contrast-enhanced CT and MRI, angiography, endoscopy, and positron emission tomography (PET), but a tumor was subsequently found in the head of the pancreas by octreotide scanning. Her diarrhea diminished dramatically after octreotide treatment, while her diarrhea has ceased without the therapy of octreotide at the first admission in the course of 2 years of her disease. Immunohistochemial analysis of the excised tumor tissue revealed the expression of both vasoactive intestinal peptide (VIP) and VIP and pituitary adenylate cyclase-activating peptide 1 (VPAC1) receptors. This is the first case report of a VIPoma that immunostains for VIP and VPAC1 receptors and indicates that abundant VIP produced by VIPoma might inhibit its growth and reduce VIP secretion via the VPAC1 receptor in vivo.

Phase Ib Trial of Oncolytic Herpes Virus G207 Shows Safety of Multiple Injections and Documents Viral Replication

The prognosis for patients with glioblastoma remains poor despite numerous phase I–II clinical trials on systemic chemotherapies such as the vascular endothelial growth factor–neutralizing antibody bevacizumab.1 Indeed, only two chemotherapies—implantable carmustine-containing Gliadel wafers and temozolomide2—have been approved for glioblastoma by the Food and Drug Administration, because these two agents showed benefit in phase III clinical trials by prolonging median survival by approximately 8 weeks. Therefore, there is a continued need to explore new treatments or to further refine and optimize older treatments for this type of brain cancer.

キャピラリー電気泳動―飛行時間型質量分析計を用いた大腸がん組織のメタボローム解析

Capillary electrophoresis-mass spectrometry based metabolomics exhibits unlimited potentials to elucidate global metabolic profiles of various types of samples including clinical tissues. In addition, non-targeted metabolome analysis by this method holds the promise of discovering novel biomarker candidates. In this commentary, we introduce our strategies for studying cancer-specific metabolism by analyzing metabolome of surgically excised tumor tissues.

Dynamic Contrast-Enhanced Derived Cerebral Blood Volume Correlates Better With Leak Correction Than With No Correction for Vascular Endothelial Growth Factor, Microvascular Density, and Grading of Astrocytoma

To look for the impact of leak correction on correlation of perfusion indices with microvessel density (MVD) and vascular endothelial growth factor (VEGF) in astrocytomas. Dynamic contrast-enhanced magnetic resonance imaging was performed in 64 patients with varying grades of astrocytoma. Perfusion indices (ie, relative cerebral blood volume (rCBV) with and without leak correction, relative cerebral blood flow (rCBF), permeability (k(trans)), and leakage (v(e)) were quantified. MVD and VEGF-expressing cells were quantified from the excised tumor tissues and were correlated with perfusion metrics. Perfusion indices showed significant difference among the astrocytoma grades. The corrected rCBV correlated better with MVD and VEGF. The corrected rCBV correlated (r = 0.853, P = <0.001) strongly, whereas the uncorrected rCBV (r = 0.592, P = <0.001) and k(trans) (r = 0.498, P = 0.001) correlated moderately with tumor grade. The corrected rCBV discriminated 100% low-grade from high-grade astrocytoma, while uncorrected rCBV did this in 95.5% low-grade and 71.4% high-grade astrocytoma. Corrected rCBV better correlates with grade and is more accurate in discriminating low-grade from high-grade astrocytoma compared with uncorrected rCBV.

Targeted therapeutic approach for an anaplastic thyroid cancer in vitro and in vivo

Anaplastic thyroid carcinoma (ATC) is among the most aggressive human malignancies, being responsible for the majority of thyroid cancer-related deaths. Despite multimodal therapy including surgery, chemotherapy, and radiotherapy, the outcome of ATC is poor. The human ATC cell line MB1, derived from tumor tissue of a 57-year-old man with thyroid cancer and pronounced neutrophilia, was established from surgically excised tumor tissue. The karyotype of the cell line shows many chromosomal abnormalities. Preclinical investigations have shown antitumor activity and effectiveness of the BRAF kinase inhibitor Sorafenib and the proteasome inhibitor Bortezomib. After establishment of the MB1 cell line these agents were applied in vitro and, showing activity in a cell culture model, were also used for in vivo treatment. Sorafenib had some clinical effect, namely normalization of leucocytosis, but had no sustained impact on subsequent tumor growth and development of distant metastasis. Molecular diagnostics of the tumor demonstrated no BRAF mutations in exons 11 and 15 concordant with a rather modest effect of Sorafenib on MB1 cell growth. Clinical benefit was seen with subsequent bortezomib therapy inducing a temporary halt to lymph node growth and a progression-free interval of 7 weeks. Our observations together with previous data from preclinical models could serve as a rationale for selecting those patients suffering from ATC most likely to benefit from targeted therapy. A prospective controlled randomized trial integrating kinase and proteasome inhibitors into a therapeutic regime for ATC is warranted.

IFN-β-Dependent Inhibition of Tumor Growth by the Vascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid (DMXAA)

By attacking established tumor vasculature, vascular disrupting agents (VDAs) represent an alternative approach to the treatment of cancer. One such VDA, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), is scheduled for phase III trials for prostate and lung cancer in combination with conventional chemotherapies. In this work, we identify interferon-beta (IFN-beta) as a central mediator in the host's response to DMXAA. In mice bearing Lewis lung adenocarcinomas, a single intraperitoneal dose of DMXAA was shown to effect a highly significant reduction in tumor growth rate in wild-type mice that was not seen in IFN-beta-null mice. Moreover, intratumoral cytokine expression was shown to be dependent on host-derived IFN-beta, as DMXAA-treated IFN-beta-null mice demonstrated a lack of induction of not only IFN-beta but also the antiangiogenic cytokine, IP-10, in excised tumor tissue. These results support the conclusion that DMXAA derives its potent anticancer properties in part through elicitation of IFN-beta expression by host-derived elements.

Inhibition of Xenograft Tumor Growth and Down-Regulation of ErbB Receptors by an Antibody Directed against Lewis Y Antigen

The blood group-related Lewis Y antigen is expressed on the majority of human cancers of epithelial origin with only limited expression on normal tissue. Therefore, the Lewis Y antigen represents an interesting candidate for antibody-based treatment strategies. Previous experiments showed that the humanized Lewis Y-specific monoclonal antibody, IGN311, reduced ErbB-receptor-mediated stimulation of mitogen-activated protein kinase by altering receptor recycling. Here, we tested whether binding of IGN311 to growth factor receptors is relevant also to inhibition of tumor growth in vivo. Prolonged incubation with IGN311 of human tumor cell lines, which express high levels of ErbB1 (A431) or ErbB2 (SK-BR-3), resulted in down-regulation of the receptors and inhibition of cell proliferation. IGN311 inhibited the growth of tumors derived from A431 cells xenografted in nude mice. Treatment with IGN311 was associated with a down-regulation of ErbB1 in the excised tumor tissue. Importantly, these effects of IGN311 were also mimicked by the Fab fragment of IGN311. These data indicate that tumor cell growth inhibition by IGN311 cannot solely be accounted for by invoking cellular and humoral immunological mechanisms. A direct effect on signaling via binding to Lewis Y glycosylated growth factor receptors on tumor cells is also likely to contribute to the therapeutic effect of IGN311 in vivo.

Giant invasive pituitary prolactinoma with falsely low serum prolactin: the significance of ‘hook effect’

The authors report a case of a patient with giant, invasive skull base tumor extending to the parasellar area discovered incidentally during the work-up for decreased memory. The patient's neurological exam was otherwise unremarkable. Endocrine evaluation performed at a local hospital showed a moderate hyperprolactinemia 103 ng/ml (normal up to 20 ng/ml). Given the large size of the tumor, the elevated prolactin (PRL) was interpreted to be secondary to stalk effect and patient underwent debulking surgery through a transcranial approach. Immunostaining of the excised tumor tissue was strongly positive for prolactin. His prolactin was found to be 13,144 ng/ml in our lab after surgery confirming the diagnosis of invasive giant prolactinoma. The patient developed a complete right third, fourth and sixth nerve palsy postoperatively. He was started on Cabergoline with normalization of his prolactin level and more than 50% decrease in residual tumor size over 9 months periods. There has been no clinically significant improvement in his right eye ophthalmoplegia since surgery. This case highlights the importance of 'Hook Effect' resulting in falsely low prolactin level, which may have significant therapeutic implication.

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is widely used as an AMP-kinase activator, which regulates energy homeostasis and response to metabolic stress. Here, we investigated the effect of AICAR, an AMPK activator, on proliferation of various cancer cells and observed that proliferation of all the examined cell lines was significantly inhibited by AICAR treatment due to arrest in S-phase accompanied with increased expression of p21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in vitro growth of cancer cells was mirrored in vivo with increased expression of p21, p27, and p53 and attenuation of Akt phosphorylation. Anti-proliferative effect of AICAR is mediated through activated AMP-activated protein kinase (AMPK) as iodotubericidin and dominant-negative AMPK expression vector reversed the AICAR-mediated growth arrest. Moreover, constitutive active AMPK arrested the cells in S-phase by inducing the expression of p21, p27, and p53 proteins and inhibiting Akt phosphorylation, suggesting the involvement of AMPK. AICAR inhibited proliferation in both LKB and LKB knock-out mouse embryo fibroblasts to similar extent and arrested cells at S-phase when transfected with dominant negative expression vector of LKB. Altogether, these results indicate that AICAR can be utilized as a therapeutic drug to inhibit cancer, and AMPK can be a potential target for treatment of various cancers independent of the functional tumor suppressor gene, LKB.

Potential of traditional ayurvedic formulation, Triphala, as a novel anticancer drug

The cytotoxic effects of aqueous extract of Triphala, an ayurvedic formulation, were investigated on human breast cancer cell line (MCF-7) and a transplantable mouse thymic lymphoma (barcl-95). The viability of treated cells was found to decrease with the increasing concentrations of Triphala. On the other hand, treatment of normal breast epithelial cells, MCF-10 F, human peripheral blood mononuclear cells, mouse liver and spleen cells, with similar concentrations of Triphala did not affect their cytotoxicity significantly. The drug treatment was found to induce apoptosis in MCF-7 and barcl-95 cells in vitro as determined by annexin-V fluorescence and proportion of apoptotic cells was found dependent on Triphala concentration. MCF-7 cells treated with Triphala when subjected to single cell gel electrophoresis, revealed a pattern of DNA damage, characteristic of apoptosis. Studies on Triphala treated MCF-7 and barcl-95 cells showed significant increase in intracellular reactive oxygen species (ROS) in a concentration dependent manner. ROS increase was, however, found to be insignificant in MCF-10 F as well as in murine spleen and liver normal cells. In vivo, direct oral feeding of Triphala to mice (40 mg/kg body weight) transplanted with barcl-95 produced significant reduction in tumor growth as evaluated by tumor volume measurement. It was also found that apoptosis was significantly higher in the excised tumor tissue of Triphala fed mice as compared to the control, suggesting the involvement of apoptosis in tumor growth reduction. These results suggest that Triphala possessed ability to induce cytotoxicity in tumor cells but spared the normal cells. The differential effect of Triphala on normal and tumor cells seems to be related to its ability to evoke differential response in intracellular ROS generation. The differential response of normal and tumor cells to Triphala in vitro and the substantial regression of transplanted tumor in mice fed with Triphala points to its potential use as an anticancer drug for clinical treatment.