Graeme J. Finlay

A semiautomated microculture method for investigating growth inhibitory effects of cytotoxic compounds on exponentially growing carcinoma cells

A method is described by which the growth inhibitory effects of cytotoxic compounds on exponentially growing adherent cell lines can be quantitated. Cultures are established in microtiter plates and the cytotoxic compounds to be tested are added after 2 days. Dilution series are constructed within the microcultures using 8-channel micropipets designed for use with microtiter plates. To measure growth after several days of drug exposure, adherent cell layers are fixed, stained, and the plates washed in water to remove excess stain. After stained cells are air-dried and solubilized, absorbance is determined using spectrophotometers designed for reading of microtiter trays. This method is reproducible and offers the advantage that any adherent cell line can be utilized without the need for tedious cell-counting procedures.

Comparison of in Vitro activity of cytotoxic drugs towards human carcinoma and leukaemia cell lines

Eight human haematopoietic cell lines and four human carcinoma lines were used to compare the activity of a number of cytotoxic drugs including amsacrine, the amsacrine analogue CI-921, methotrexate, nitracrine, doxorubicin, daunorubicin and 5-fluorouracil. Activity was assessed by means of semiautomated microculture growth inhibition assays. Cell density of the non-adherent cell lines was measured using the technique of Mosmann (J Immunol Methods 1983, 65, 55-63), in which the dye thiazolyl blue (MTT) is metabolised to a dark blue formazan product. This technique gives similar results to those obtained by direct cell counting in an electronic cell counter, and when applied to some adherent cell lines gives similar results to those obtained by the methylene blue staining technique previously developed (Anal Biochem 1984, 139, 272-277). Both methylene blue and MTT methods were used to investigate cytotoxicity in conjunction with semi-automated 96-well microculture plate techniques. The results show that the three T-cell leukaemia lines (CCRF-CEM, Jurkat and MOLT-4) are more sensitive to DNA-binding drugs (excluding nitracrine) than are the colon carcinoma lines (HCT-8, HT-29, SW480 and SW620). The more resistant haematopoietic lines are intermediate in drug sensitivity between the T cell leukaemia and carcinoma lines. The DNA binding drugs show remarkably similar patterns of differential activity against the different cell lines.

Signaling Pathways in Melanogenesis

Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis.

Synthesis and antitumor activity of some indeno[1,2-b]quinoline-based bis carboxamides.

A series of bis(11-oxo-11H-indeno[1,2-b]quinoline-6-carboxamides) linked through the 6-carboxamides were prepared by coupling the requisite acid imidazolides with various diamines. Compounds with mono-cationic linker chains were more potent cytotoxins than the corresponding monomer in a panel of rodent and human cell lines, while those with the dicationic linker chains (CH2)2NR(CH2)2NR(CH2)2 and (CH2)2NR(CH2)3NR(CH2)2 showed extraordinarily high potencies (for example, IC50s of 0.18-1.4 nM against human Jurkat leukemia; up to 1000-fold more potent than the parent monomer). As seen previously in the monomeric series, small, lipophilic 4-substituents significantly increased potency in cell culture. The dimeric compounds were all slightly to significantly more potent in the mutant JL(A) and JL(D) cell lines that under-express topo II, suggesting that this enzyme is not their primary target. An 11-imino-linked dimer was much less active, and an asymmetric indeno[1,2-b]quinoline-6-carboxamide/naphthalimide dimer was less active than the comparable symmetric bis(indeno[1,2-b]quinoline-6-carboxamide). Selected analogues were active against sub-cutaneously implanted colon 38 tumors in mice, giving growth delays comparable to that of the clinical topo I inhibitor irinotecan at up to 10-fold lower doses. These compounds form an interesting new class of putative topo I inhibitors.

Epigenetic regulation in human melanoma: past and future.

The development and progression of melanoma have been attributed to independent or combined genetic and epigenetic events. There has been remarkable progress in understanding melanoma pathogenesis in terms of genetic alterations. However, recent studies have revealed a complex involvement of epigenetic mechanisms in the regulation of gene expression, including methylation, chromatin modification and remodeling, and the diverse activities of non-coding RNAs. The roles of gene methylation and miRNAs have been relatively well studied in melanoma, but other studies have shown that changes in chromatin status and in the differential expression of long non-coding RNAs can lead to altered regulation of key genes. Taken together, they affect the functioning of signaling pathways that influence each other, intersect, and form networks in which local perturbations disturb the activity of the whole system. Here, we focus on how epigenetic events intertwine with these pathways and contribute to the molecular pathogenesis of melanoma.

Emerging Role of Long Non-Coding RNA SOX2OT in SOX2 Regulation in Breast Cancer

The transcription factor SOX2 is essential for maintaining pluripotency in a variety of stem cells. It has important functions during embryonic development, is involved in cancer stem cell maintenance, and is often deregulated in cancer. The mechanism of SOX2 regulation has yet to be clarified, but the SOX2 gene lies in an intron of a long multi-exon non-coding RNA called SOX2 overlapping transcript (SOX2OT). Here, we show that the expression of SOX2 and SOX2OT is concordant in breast cancer, differentially expressed in estrogen receptor positive and negative breast cancer samples and that both are up-regulated in suspension culture conditions that favor growth of stem cell phenotypes. Importantly, ectopic expression of SOX2OT led to an almost 20-fold increase in SOX2 expression, together with a reduced proliferation and increased breast cancer cell anchorage-independent growth. We propose that SOX2OT plays a key role in the induction and/or maintenance of SOX2 expression in breast cancer.

Comparison of the effects of the PI3K/mTOR inhibitors NVP-BEZ235 and GSK2126458 on tamoxifen-resistant breast cancer cells

Background: Treatment with anti-estrogens or aromatase inhibitors is commonly used for patients with estrogen receptor-positive (ER+) breast cancers; however resistant disease develops almost inevitably, requiring a choice of secondary therapy. One possibility is to use inhibitors of the PI3K/mTOR pathway and several candidate drugs are in development. We examined the in vitro effects of two inhibitors of the PI3K/mTOR pathway on resistant MCF-7 cells. Methods: We cultured MCF-7 cells for prolonged periods either in the presence of the anti-estrogen tamoxifen (3 sub-lines) or in estrogen free medium (2 sub-lines) to mimic the effects of clinical treatment. We then analyzed the effects of two dual PI3K/mTOR phosphoinositide-3-kinase inhibitors, NVP-BEZ235 and GSK2126458, on the growth and signaling pathways of these MCF-7 sub-lines. The functional status of the PI3K, mTOR and ERK pathways was analyzed by measuring phosphorylation of AKT, p70S6K, rpS6 and ERK. Results: The derived sub-lines showed increased resistance to tamoxifen but none exhibited concomitantly increased sensitivity to the PI3K inhibitors. NVP-BEZ235 and GSK2126458 acted mainly by induction of cell cycle arrest, particularly in G1-phase, rather than by induction of apoptosis. The lines varied considerably in their utilization of the AKT, p70S6K and ERK pathways. NVP-BEZ235 and GSK2126458 inhibited AKT signaling but NVP-BEZ235 showed greater effects than GSK2126458 on p70S6K and rpS6 signaling with effects resembling those of rapamycin. Conclusion: Increased resistance to tamoxifen in these MCF-7 sub-lines is not associated with hypersensitivity to PI3K inhibitors. While both drugs inhibited AKT signaling, NVP-BEZ235 resembled rapamycin in inhibiting the mTOR pathway. See commentary: Overcoming resistance: Targeting the PI3K/mTOR pathway in endocrine refractory breast cancer

Effects of protein binding on the in vitro activity of antitumour acridine derivatives and related anticancer drugs.

Purpose: We set out to measure drug binding to serum proteins. These have been shown to reduce the free plasma concentrations of a number of anticancer drugs, particularly of those of complex organic structure, in both experimental studies and clinical trials. Methods: We have used cultures of murine Lewis lung carcinoma cells as sensors of available drug to measure the effects of two drug-binding plasma proteins, α-acid glycoprotein (AAG) and human serum albumin (HSA), as well as of bovine serum albumin (BSA) on drug activity. Results: The concentrations required for 50% growth inhibition (IC50 values) of a number of anticancer drugs were found to be linear functions of the added proteins. Assuming that cells respond to free drug, the data provide estimates of the product K · n, where K is the binding constant of the protein and n is the number of drug binding sites per protein molecule. Amsacrine, the amsacrine analogue asulacrine, camptothecin, DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide), doxorubicin, etoposide, mitoxantrone, paclitaxel and vincristine were tested. The K · n values for AAG were 30, 2400, 8.7, 340, 29, 290 × 103 M−1 and 120 × 103 M−1, respectively, and the K · n values for HSA were 16, 580, 530, 10, 6.2, 4.3 × 103 M−1 and 0.0 × 103 M−1, respectively. The combined data allowed the estimation of free fractions of drug in plasma, assuming that AAG and HSA contributed most to protein binding. The data were in general comparable with that reported using equilibrium dialysis and ultrafiltration. Data for drug binding to BSA were different from those for HSA, in some cases by a large factor with values for HSA generally higher. The applicability of the method to analogue development was illustrated by examining the binding to AAG of a series of DACA analogues, and binding was found to be primarily related to lipophilicity. Conclusion: IC50 determinations provide a rapid means of estimating drug binding to plasma proteins and have utility in the assessment of new anticancer drugs.

In vitro assessment ofN-[2-(dimethylamino)ethyl]acridine-4-carboxamide, a DNA-intercalating antitumour drug with reduced sensitivity to multidrug resistance

SummaryThe successful treatment of cancer requires the identification of new drugs with novel actions.N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide dihydrochloride (DACA) is a topoisomerase II-targeted antitumour drug with curative activity against murine Lewis lung carcinoma. DACA was assessed for novel patterns of growth inhibition using normal and multidrug-resistant human cell lines. Cells were cultured in 96-well microtitre trays and tested against DACA and related topoisomerase-directed drugs, including amsacrine, etoposide and doxorubicin, and drug concentrations for 50% growth inhibition (IC50 or GI50 values) were determined. In a series of Jurkat leukaemia lines characterised as exhibiting atypical multidrug resistance, DACA was to a large extent capable of overcoming multidrug resistance exhibited towards the other topoisomerase-directed agents. DACA was also tested against the National Cancer Institute 60-tumour-specific cell-line panel (GI50 values ranging from 420 to 5,400 nM; mean, 2,100 nM) and against a series of primary cultures of surgically excised melanomas (IC50 values ranging from 60 to 1,600 nM; mean, 590 nM). DELTA values (deviations of logarithmic IC50 or GI50 values from the mean) were calculated and compared by correlation analysis. The standard deviation of DELTA values was found to be lower for DACA than for the other topoisomerase II-directed drugs amsacrine, etoposide, doxorubicin and mitozantrone in both the cell lines and the primary cultures. These lower standard deviations appear to have resulted from the reduced susceptibility of DACA to both P-glycoprotein- and topoisomerase II-mediated multidrugresistance mechanisms occurring naturally in cell lines and primary cultures.

Positioning of the carboxamide side chain in 11-oxo-11H-indeno[1,2-b]quinolinecarboxamide anticancer agents: effects on cytotoxicity.

A series of 11-oxo-11H-indeno[1,2-b]quinolines bearing a carboxamide-linked cationic side chain at various positions on the chromophore was studied to determine structure-activity relationships between cytotoxicity and the position of the side chain. The compounds were prepared by Pfitzinger synthesis from an appropriate isatin and 1-indanone, followed by various oxidative steps, to generate the required carboxylic acids. The 4- and 6-carboxamides (with the side chain on a terminal ring, off the short axis of the chromophore) were effective cytotoxins. The dimeric 4- and 6-linked analogues were considerably more cytotoxic than the parent monomers, but had broadly similar activities. In contrast, analogues with side chains at the 8-position (on a terminal ring but off the long axis of the chromophore) or 10-position (off the short axis of the chromophore but in a central ring) were drastically less effective. The 4,10- and 6,10-biscarboxamides had activities between those of the corresponding parent monocarboxamides. The first of these showed good activity against advanced subcutaneous colon 38 tumours in mice.