Passage number of cancer cell lines: Importance, intricacies, and way-forward.

Cancer cell lines play a crucial role as invaluable models in cancer research, facilitating the examination of cancer progression as well as the advancement of diagnostics and treatments. While they may not perfectly replicate the original tumor, they generally exhibit similar characteristics. Low-passage cancer cell lines are generally preferred due to their closer resemblance to the original tumor, as long-term culturing can alter the genetic and molecular profiles of a cell line thereby highlighting the importance of monitoring the passage number (PN). Variations in proliferation, migration, gene expression, and drug sensitivity can be linked to PN differences. PN can also influence DNA methylation levels, metabolic profiles, and the expression of genes/or proteins in cancer cell lines. When conducting research on cancer cell lines, it is crucial for researchers to carefully select the appropriate PN to maintain consistency and reliability of results. Moreover, to ensure dependability and replicability, scientists ought to actively track the growth, migration, and gene/or protein profiles of cancer cell lines at specific PNs. This approach enables the identification of the most suitable range of PNs for experiments, guaranteeing consistent and precise results. Additionally, such efforts serve to minimize disparities and uphold the integrity of research. In this review, we have laid out recommendations for laboratories to overcome these PN discrepancies when working with cancer cell lines.

Increased MARCKS Activity in BRAF Inhibitor-Resistant Melanoma Cells Is Essential for Their Enhanced Metastatic Behavior Independent of Elevated WNT5A and IL-6 Signaling.

Treatment of melanoma with a BRAF inhibitor (BRAFi) frequently initiates development of BRAFi resistance, leading to increased tumor progression and metastasis. Previously, we showed that combined inhibition of elevated WNT5A and IL-6 signaling reduced the invasion and migration of BRAFi-resistant (BRAFi-R) melanoma cells. However, the use of a combined approach per se and the need for high inhibitor concentrations to achieve this effect indicate a need for an alternative and single target. One such target could be myristoylated alanine-rich C-kinase substrate (MARCKS), a downstream target of WNT5A in BRAFi-sensitive melanoma cells. Our results revealed that MARCKS protein expression and activity are significantly elevated in PLX4032 and PLX4720 BRAFi-R A375 and HTB63 melanoma cells. Surprisingly, neither WNT5A nor IL-6 contributed to the increases in MARCKS expression and activity in BRAFi-R melanoma cells, unlike in BRAFi-sensitive melanoma cells. However, despite the above findings, our functional validation experiments revealed that MARCKS is essential for the increased metastatic behavior of BRAFi-R melanoma cells. Knockdown of MARCKS in BRAFi-R melanoma cells caused reductions in the F-actin content and the number of filopodia-like protrusions, explaining the impaired migration, invasion and metastasis of these cells observed in vitro and in an in vivo zebrafish model. In our search for an alternative explanation for the increased activity of MARCKS in BRAFi-R melanoma cells, we found elevated basal activities of PKCα, PKCε, PKCι, and RhoA. Interestingly, combined inhibition of basal PKC and RhoA effectively impaired MARCKS activity in BRAFi-R melanoma cells. Our results reveal that MARCKS is an attractive single antimetastatic target in BRAFi-R melanoma cells.

Metabolic changes in triple negative breast cancer-focus on aerobic glycolysis.

Among breast cancer subtypes, the triple negative breast cancer (TNBC) has the worst prognosis. In absence of any permitted targeted therapy, standard chemotherapy is the mainstay for TNBC treatment. Hence, there is a crucial need to identify potential druggable targets in TNBCs for its effective treatment. In recent times, metabolic reprogramming has emerged as cancer cells hallmark, wherein cancer cells display discrete metabolic phenotypes to fuel cell progression and metastasis. Altered glycolysis is one such phenotype, in which even in oxygen abundance majority of cancer cells harvest considerable amount of energy through elevated glycolytic-flux. In the present review, we attempt to summarize the role of key glycolytic enzymes i.e. HK, Hexokinase; PFK, Phosphofructokinase; PKM2, Pyruvate kinase isozyme type 2; and LDH, Lactate dehydrogenase in TNBCs, and possible therapeutic options presently available.

WNT5A-Induced Activation of the Protein Kinase C Substrate MARCKS Is Required for Melanoma Cell Invasion.

WNT5A is a well-known mediator of melanoma cell invasion and metastasis via its ability to activate protein kinase C (PKC), which is monitored by phosphorylation of the endogenous PKC substrate myristoylated alanine-rich c-kinase substrate (MARCKS). However, a possible direct contribution of MARCKS in WNT5A-mediated melanoma cell invasion has not been investigated. Analyses of melanoma patient databases suggested that similar to WNT5A expression, MARCKS expression appears to be associated with increased metastasis. A relationship between the two is suggested by the findings that recombinant WNT5A (rWNT5A) induces both increased expression and phosphorylation of MARCKS, whereas WNT5A silencing does the opposite. Moreover, WNT5A-induced invasion of melanoma cells was blocked by siRNA targeting MARCKS, indicating a crucial role of MARCKS expression and/or its phosphorylation. Next, we employed a peptide inhibitor of MARCKS phosphorylation that did not affect MARCKS expression and found that it abolished WNT5A-induced melanoma cell invasion. Similarly, rWNT5A induced the accumulation of phosphorylated MARCKS in membrane protrusions at the leading edge of melanoma cells. Our results demonstrate that WNT5A-induced phosphorylation of MARCKS is not only an indicator of PKC activity but also a crucial regulator of the metastatic behavior of melanoma and therefore an attractive future antimetastatic target in melanoma patients.

Combination therapy targeting the elevated interleukin-6 level reduces invasive migration of BRAF inhibitor-resistant melanoma cells.

The identification of novel antimetastatic therapeutic targets is necessary for improved treatment of patients with acquired BRAF inhibitor-resistant (BRAFi-R) melanoma, in whom metastasis is a major concern. Our present study focused on the identification of such targets to explore novel antimetastatic therapeutic options for BRAFi-R melanoma patients. We confirmed the development of BRAFi resistance in our BRAFi-treated melanoma cell lines by demonstrating reduced sensitivity to BRAF inhibitors, increased ERK1/2 activity and increased WNT5A expression. Here, we demonstrated for the first time that high secretion of interleukin-6 (IL-6) was associated with increased invasive migration of BRAFi-R melanoma cells. This finding could be readily explained by the increased expression of WNT5A in BRAFi-R melanoma cells and the presence of an IL-6/WNT5A positive feedback loop in parental melanoma cells. Surprisingly, however, we found that the IL-6/WNT5A positive feedback loop present in parental melanoma cells was lost during the development of acquired BRAFi resistance, meaning that IL-6 and WNT5A signalling were independent events in BRAFi-R melanoma cells. Despite the absence of an IL-6/WNT5A loop, we found that both an IL-6 blocking antibody and the WNT5A antagonist Box5 alone impaired the elevated invasive migration of BRAFi-R melanoma cells, but combined use of the two was more effective. This impaired invasive migration of BRAFi-R melanoma cells correlated well with the reduction in Cdc42-GTPase activity and alterations of the actin cytoskeleton in these cells. In summary, our novel identification of IL-6 as a key independent promoter of the invasive migration of BRAFi-R melanoma cells stresses that a combination of a blocking IL-6 antibody and administration of the WNT5A antagonist Box5 might be an attractive antimetastatic approach for future treatment of BRAFi-R melanoma patients.

WNT5A as a therapeutic target in breast cancer.

Despite the clinical development of novel adjuvant and neoadjuvant chemotherapeutic drugs, metastatic breast cancer is one of the leading causes of cancer-related death among women. The present review focuses on the relevance, mechanisms, and therapeutic potential of targeting WNT5A as a future anti-metastatic treatment strategy for breast cancer patients by restoring WNT5A signaling as an innovative therapeutic option. WNT5A is an auto- and paracrine ß-catenin-independent ligand that has been shown to induce tumor suppression as well as oncogenic signaling, depending upon cancer type. In breast cancer patients, WNT5A protein expression has been observed to be significantly reduced in between 45 and 75% of the cases and associated with early relapse and reduced disease-free survival. WNT5A triggers various downstream signaling pathways in breast cancer that primarily affect tumor cell migration and invasion. The accumulated in vitro results reveal that treatment of WNT5A-negative breast cancer cells with recombinant WNT5A caused different tumor-suppressive responses and in particular it impaired migration and invasion. The anti-migratory/invasive and anti-metastatic effects of reconstituting WNT5A signaling by the small WNT5A mimicking peptide Foxy5 form the basis for two successful clinical phase 1-studies aiming at determining safety and pharmacokinetics as well as defining dose-level for a subsequent phase 2-study. We conclude that re-installation of WNT5A signaling is an attractive and promising anti-metastatic therapeutic approach for future treatment of WNT5A-negative breast cancer patients.

Demonstration of a WNT5A-IL-6 positive feedback loop in melanoma cells: Dual interference of this loop more effectively impairs melanoma cell invasion.

Increased expression and signalling of WNT5A and interleukin-6 (IL-6) have both been shown to promote melanoma progression. Here, we investigated the proposed existence of a WNT5A-IL-6 positive feedback loop that drives melanoma migration and invasion. First, the HOPP algorithm revealed that the invasive phenotype of cultured melanoma cells was significantly correlated with increased expression of WNT5A or IL-6. In three invasive melanoma cell lines, endogenous WNT5A protein expression was related to IL-6 protein secretion. Knockdown with anti-IL-6 siRNAs or treating WM852 melanoma cells with a neutralising anti-IL-6 antibody reduced WNT5A protein expression. Conversely, the silencing of WNT5A expression by WNT5A siRNAs or treating WM852 melanoma cells with Box5 (a WNT5A antagonist) significantly reduced IL-6 secretion. Interestingly, these effects occurred at the protein level but not at the transcriptional levels. Functionally, we demonstrated that combined siRNA knockdown of WNT5A and IL-6 expression or the simultaneous inhibition of WNT5A and IL-6 signalling inhibited melanoma cell invasion more effectively than suppressing each factor individually. Together, our results demonstrate that WNT5A and IL-6 are connected through a positive feedback loop in melanoma cells and that the combined targeting of both molecules could serve as an effective therapeutic means to reduce melanoma metastasis.

Therapy for BRAFi-Resistant Melanomas: Is WNT5A the Answer?

In recent years, scientists have advocated the use of targeted therapies in the form of drugs that modulate genes and proteins that are directly associated with cancer progression and metastasis. Malignant melanoma is a dreadful cancer type that has been associated with the rapid dissemination of primary tumors to multiple sites, including bone, brain, liver and lungs. The discovery that approximately 40%-50% of malignant melanomas contain a mutation in BRAF at codon 600 gave scientists a new approach to tackle this disease. However, clinical studies on patients have shown that although BRAFi (BRAF inhibitors) trigger early anti-tumor responses, the majority of patients later develop resistance to the therapy. Recent studies have shown that WNT5A plays a key role in enhancing the resistance of melanoma cells to BRAFi. The focus of the current review will be on melanoma development, signaling pathways important to acquired resistance to BRAFi, and why WNT5A inhibitors are attractive candidates to be included in combinatorial therapies for melanoma.

Resveratrol and curcumin synergistically induces apoptosis in cigarette smoke condensate transformed breast epithelial cells through a p21(Waf1/Cip1) mediated inhibition of Hh-Gli signaling.

Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggressive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of curcumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC50 value was noted in cells treated with the combination of resveratrol (3µM) and curcumin (3µM) in comparison to 30µM of resveratrol or curcumin alone. Resveratrol+curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, ß-catenin and cleaved notch-1 val1744, respectively. Furthermore, the combination also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21(Waf/Cip1) in vitro and in vivo. A significant reduction of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21(Waf/Cip1) knockout cells suggests this combination caused apoptosis through p21(Waf/Cip1). Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2(Waf/Cip1) mediated inhibition of Hedgehog-Gli cascade.

Pentacyclic Triterpenoids Inhibit IKKß Mediated Activation of NF-κB Pathway: In Silico and In Vitro Evidences.

Pentacyclic Triterpenoids (PTs) and their analogues as well as derivatives are emerging as important drug leads for various diseases. They act through a variety of mechanisms and a majority of them inhibit the nuclear factor kappa-beta (NF-κB) signaling pathway. In this study, we examined the effects of the naturally occurring PTs on IκB kinase-ß (IKKß), which has great scientific relevance in the NF-κB signaling pathway. On virtual screening, 109 PTs were screened through the PASS (prediction of activity spectra of substances) software for prediction of NF-κB inhibitory activity followed by docking on the NEMO/IKKß association complex (PDB: 3BRV) and testing for compliance with the softened Lipinski's Rule of Five using Schrodinger (LLC, New York, USA). Out of the projected 45 druggable PTs, Corosolic Acid (CA), Asiatic Acid (AA) and Ursolic Acid (UA) were assayed for IKKß kinase activity in the cell free medium. The UA exhibited a potent IKKß inhibitory effect on the hotspot kinase assay with IC50 of 69 µM. Whereas, CA at 50 µM concentration markedly reduced the NF-κB luciferase activity and phospho-IKKß protein expressions. The PTs tested, attenuated the expression of the NF-κB cascade proteins in the LPS-stimulated RAW 264.7 cells, prevented the phosphorylation of the IKKα/ß and blocked the activation of the Interferon-gamma (IFN-γ). The results suggest that the IKKß inhibition is the major mechanism of the PTs-induced NF-κB inhibition. PASS predictions along with in-silico docking against the NEMO/IKKß can be successfully applied in the selection of the prospective NF-κB inhibitory downregulators of IKKß phosphorylation.

The Apoptotic Effect of Plant Based Nanosilver in Colon Cancer Cells is a p53 Dependent Process Involving ROS and JNK Cascade.

Here, we report the p53 dependent mitochondria-mediated apoptotic mechanism of plant derived silver-nanoparticle (PD-AgNPs) in colorectal cancer cells (CRCs). PD-AgNPs was synthesized by reduction of AgNO3 with leaf extract of a medicinal plant periwinkle and characterized. Uptake of PD-AgNPs (ξ - 2.52 ± 4.31 mV) in HCT116 cells was 3 fold higher in comparison to synthetic AgNPs (ξ +2.293 ± 5.1 mV). A dose dependent increase in ROS production, activated JNK and decreased mitochondrial membrane potential (MMP) were noted in HCT116 but not in HCT116 p53(-/-) cells after PD-AgNP exposure. PD-AgNP-mediated apoptosis in CRCs is a p53 dependent process involving ROS and JNK cascade.

5-Fluorouracil mediated anti-cancer activity in colon cancer cells is through the induction of Adenomatous Polyposis Coli: Implication of the long-patch base excision repair pathway.

Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLß and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.

Synthesis and biological evaluation of andrographolide analogues as anti-cancer agents.

A new family of andrographolide analogues were synthesized and screened in vitro against kidney (HEK-293) and breast (MCF-7) cancer cells. The anti-cancer effects of the active analogues (2b, 2c and 4c) were determined by multiple cell based assays such as MTT, immunostaining, FACS, western blotting and transcriptional inhibition of NF-κB activity. Importantly, these compounds were found to possess higher anti-cancer potency than andrographolide and low toxicity to normal (VERO and MCF-10A) cells. Increased level of Bax/Bcl-xL ratio, caspase 3, and sub G1 population, higher expression level of tumor suppressor protein p53 and lower expression level of NF-κB suggested potent apoptotic property of the active analogues. Data revealed that the andrographolide derivative-mediated cell death in cancer cells was p53 dependent.

Cytotoxic effect of microbial biosurfactants against human embryonic kidney cancerous cell: HEK-293 and their possible role in apoptosis.

Two different microbial biosurfactants S9BS and CHBS were isolated from Lysinibacillus fusiformis S9 and Bacillus tequilensis CH. Cytotoxicity effect of these biosurfactants on human embryonic kidney cancerous cell (HEK-293) were studied with the help of 3-(4,5-dimethylthiazol-2yl-)-2, 5-diphenyl tetrazolium bromide (MTT) assay and morphological changes were observed under inverted microscope. The biosurfactants exhibited positive cytotoxic effect on HEK-293 cell line. It was found that LC50 of S9BS and CHBS were 75 and 100 µg ml(-1), respectively. Further cell cycle and apoptosis analysis of biosurfactant-treated HEK-293 cell line were done by FACS. In this study, cytotoxic effect of glycolipid biosurfactant against HEK-293 cell lines is reported for the first time. Mechanism towards increased membrane permeability of biosurfactant-treated cancer cell may be the incorporation of its lipid moiety into the plasma membrane leading to formation of pores and membrane disruption. Hence, these microbial biosurfactants can prove to be significant biomolecule for cancer treatment.

Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway.

Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-ß, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21.

The contribution of heavy metals in cigarette smoke condensate to malignant transformation of breast epithelial cells and in vivo initiation of neoplasia through induction of a PI3K-AKT-NFκB cascade.

Cigarette smoking is a crucial factor in the development and progression of multiple cancers including breast. Here, we report that repeated exposure to a fixed, low dose of cigarette smoke condensate (CSC) prepared from Indian cigarettes is capable of transforming normal breast epithelial cells, MCF-10A, and delineate the biochemical basis for cellular transformation. CSC transformed cells (MCF-10A-Tr) were capable of anchorage-independent growth, and their anchorage dependent growth and colony forming ability were higher compared to the non-transformed MCF-10A cells. Increased expression of biomarkers representative of oncogenic transformation (NRP-1, Nectin-4), and anti-apoptotic markers (PI3K, AKT, NFκB) were also noted in the MCF-10A-Tr cells. Short tandem repeat (STR) profiling of MCF-10A and MCF-10A-Tr cells revealed that transformed cells acquired allelic variation during transformation, and had become genetically distinct. MCF-10A-Tr cells formed solid tumors when implanted into the mammary fat pads of Balb/c mice. Data revealed that CSC contained approximately 1.011µg Cd per cigarette equivalent, and Cd (0.0003µg Cd/1×10(7) cells) was also detected in the lysates from MCF-10A cells treated with 25µg/mL CSC. In similar manner to CSC, CdCl2 treatment in MCF-10A cells caused anchorage independent colony growth, higher expression of oncogenic proteins and increased PI3K-AKT-NFκB protein expression. An increase in the expression of PI3K-AKT-NFκB was also noted in the mice xenografts. Interestingly, it was noted that CSC and CdCl2 treatment in MCF-10A cells increased ROS. Collectively, results suggest that heavy metals present in cigarettes of Indian origin may substantially contribute to tumorigenesis by inducing intercellular ROS accumulation and increased expression of PI3K, AKT and NFκB proteins.

Induction of apoptosis by 4-(3-(tert-butylamino)imidazo[1,2-α]pyridine-2-yl) benzoic acid in breast cancer cells via upregulation of PTEN.

We have previously reported that 4-(3-(tert-butylamino)imidazo[1,2-α]pyridine-2-yl)benzoic acid, a bicyclic N-fused aminoimidazoles derivative (BNFA-D), possesses anticancer potentiality against breast and kidney cancer cells with minimal toxicities to corresponding normal cells. Here, we explored the mechanism of action of BNFA-D in breast cancer cells using multiple cell-based assays such as MTT, DAPI, FACS, Western blot, and immunoprecipitation. BNFA-D caused apoptosis by upregulating PTEN leading to inhibition of Wnt/TCF signaling cascade and arresting S phase in breast cancer cells. Expression levels of ß-catenin, cyclin D1, C-MYC, and phospho-AKT (Ser(473)) decreased with simultaneous increase in the levels of GSK3ß, CK1, and PTEN in BNFA-D-treated MCF-7 cells. Interestingly, silencing of PTEN in breast cancer cells reversed the phenomenon of Wnt/TCF signaling cascade inhibition after BNFA-D treatment.

1,3-Bis(2-chloroethyl)-1-nitrosourea enhances the inhibitory effect of resveratrol on 5-fluorouracil sensitive/resistant colon cancer cells.

AIM: To study the mechanism of 5-fluorouracil (5-FU) resistance in colon cancer cells and to develop strategies for overcoming such resistance by combination treatment. METHODS: We established and characterized a 5-FU resistance (5-FU-R) cell line derived from continuous exposure (25 µmol/L) to 5-FU for 20 wk in 5-FU sensitive HCT-116 cells. The proliferation and expression of different representative apoptosis and anti-apoptosis markers in 5-FU sensitive and 5-FU resistance cells were measured by the MTT assay and by Western blotting, respectively, after treatment with Resveratrol (Res) and/or 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU). Apoptosis and cell cycle arrest was measured by 4',6'-diamidino-2-phenylindole hydrochloride staining and fluorescence-activated cell sorting analysis, respectively. The extent of DNA damage was measured by the Comet assay. We measured the visible changes in the DNA damage/repair cascade by Western blotting. RESULTS: The widely used chemotherapeutic agents BCNU and Res decreased the growth of 5-FU sensitive HCT-116 cells in a dose dependent manner. Combined application of BCNU and Res caused more apoptosis in 5-FU sensitive cells in comparison to individual treatment. In addition, the combined application of BCNU and Res caused a significant decrease of major DNA base excision repair components in 5-FU sensitive cells. We established a 5-FU resistance cell line (5-FU-R) from 5-FU-sensitive HCT-116 (mismatch repair deficient) cells that was not resistant to other chemotherapeutic agents (e.g., BCNU, Res) except 5-FU. The 5-FU resistance of 5-FU-R cells was assessed by exposure to increasing concentrations of 5-FU followed by the MTT assay. There was no significant cell death noted in 5-FU-R cells in comparison to 5-FU sensitive cells after 5-FU treatment. This resistant cell line overexpressed anti-apoptotic [e.g., AKT, nuclear factor κB, FLICE-like inhibitory protein), DNA repair (e.g., DNA polymerase beta (POL-ß), DNA polymerase eta (POLH), protein Flap endonuclease 1 (FEN1), DNA damage-binding protein 2 (DDB2)] and 5-FU-resistance proteins (thymidylate synthase) but under expressed pro-apoptotic proteins (e.g., DAB2, CK1) in comparison to the parental cells. Increased genotoxicity and apoptosis were observed in resistant cells after combined application of BCNU and Res in comparison to untreated or parental cells. BCNU increased the sensitivity to Res of 5-FU resistant cells compared with parental cells. Fifty percent cell death were noted in parental cells when 18 µmol/L of Res was associated with fixed concentration (20 µmol/L) of BCNU, but a much lower concentration of Res (8 µmol/L) was needed to achieve the same effect in 5-FU resistant cells. Interestingly, increased levels of adenomatous polyposis coli and decreased levels POL-ß, POLH, FEN1 and DDB2 were noted after the same combined treatment in resistant cells. CONCLUSION: BCNU combined with Res exerts a synergistic effect that may prove useful for the treatment of colon cancer and to overcome drug resistance.

Structural elaboration of a natural product: identification of 3,3'-diindolylmethane aminophosphonate and urea derivatives as potent anticancer agents.

An approach involving rational structural elaboration of the biologically active natural product diindolylmethane (DIM) with the incorporation of aminophosphonate and urea moieties toward the discovery of potent anticancer agents was considered. A four-step approach for the synthesis of DIM aminophosphonate and urea derivatives was established. These novel compounds showed potent anticancer activities in two representative kidney and colon cancer cell lines, low toxicity to normal cells, higher potency than the parent natural product DIM and etoposide, and potent inhibition of cancer cell migration. Biophysical and immunological studies, including DAPI nuclear staining, western blot analysis with apoptotic protein markers, flow cytometry, immunocytochemistry, and comet assays of the two most potent compounds revealed good efficacies in apoptosis and DNA damage. It was found that down-regulation of nuclear factor κB (NF-κB p65) could be an important mode of action in apoptosis, and the two most potent derivatives were found to be more potent than parent compound DIM in the down-regulation of NF-κB. Our results show the importance of structural elaboration of DIM by rational incorporation of aminophosphonate and urea moieties to produce potent anticancer agents; they also suggest that this approach using other structurally simple bioactive natural products as scaffolds holds promise for future drug discovery and development.

Silver-based nanoparticles induce apoptosis in human colon cancer cells mediated through p53.

AIM: The authors have systematically investigated the anticancer potentiality of silver-based nanoparticles (AgNPs) and the mechanism underlying their biological activity in human colon cancer cells. MATERIALS & METHODS: Starch-capped AgNPs were synthesized, characterized and their biological activity evaluated through multiple biochemical assays. RESULTS: AgNPs decreased the growth and viability of HCT116 colon cancer cells. AgNP exposure increased apoptosis, as demonstrated by an increase in 4´,6-diamidino-2-phenylindole-stained apoptotic nuclei, BAX/BCL-XL ratio, cleaved poly(ADP-ribose) polymerase, p53, p21 and caspases 3, 8 and 9, and by a decrease in the levels of AKT and NF-κB. The cell population in the G1 phase decreased, and the S-phase population increased after AgNP treatment. AgNPs caused DNA damage and reduced the interaction between p53 and NF-κB. Interestingly, no significant alteration was noted in the levels of p21, BAX/BCL-XL and NF-κB after AgNP treatment in a p53-knockout HCT116 cell line. CONCLUSION: AgNPs are bona fide anticancer agents that act in a p53-dependent manner. Original submitted 16 March 2012; Revised submitted 25 August 2012; Published online 21 March 2013.