Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents) - Volume 14, Issue 8, 2014

“There is no satisfactory treatment of glioblastoma multiforme of the brain” was the introductory sentence of an article published in 1967 by Jelsma & Bucy [1]. Unfortunately this poor prognosis has not changed during the last 45 years and this pessimistic statement remains largely valid. At that time the 5 years survival was less than 10%. A historical comparison of Kaplan-Meier survival plots made in 2000 by Dr. Eric Holland showed that about 90% of patients died within 5 years. Due to the poor prognosis of patients GBM has been called “the Terminator“ [2]. Since its introduction in 2005, temozolomide (TMZ) has become the standard treatment despite the fact that the 5 years survival rate modestly increased around 20% [3]. As in 2010, TMZ in selected groups of patients had limited benefit [4]. Kast et al, pointed out that the 22 clinical trials reported in 2012 using drugs that showed promising potential in preclinical studies were found to minimally improve quality of life and overall survival when compared to the “Stupp protocol” [5]. In this hot topic issue we highlight few exciting areas that may provide useful ways to understand the complexity of glioma biology and its implication for the development of novel effective therapeutic strategies. Like in other cancers, the isolation of putative cancer stem cells (GSCs) in glioma cell lines and fresh specimens were received with great enthusiasm. The review by Alexandru et al “Selective Therapeutic Targeting of Malignant Glioma Stem Cells” [6] discusses the current therapeutic modalities for glioma treatment and highlights the potential underlying mechanism(s) for resistance to radiation and TMZ attributed to the presence of GSCs. Later on, the authors provide a list of potential targets that could be exploited to overcome the inherent resistance of GSCs. The list included microenvironemental factors (hypoxia), signaling pathways (Notch, Hedgehog – GLI, Epidermal growth factor receptor (EGFR), transforming growth factor beta (TGFβ), platelet derived growth factor receptor (PDGFR)), the c-Myc oncogene, the Bmi1 epigenetic silencer gene, overexpression of chemokine receptors, adhesion molecules, bone morphogenic protein (BMP), the stem cell marker CD133, miRNA and potential targets to overcome GSC immune surveillance escape. Some of these promising targets are further discussed in other articles in this hot topic issue. The article “Microenvironment and Brain Tumor Stem Cell Maintenance: Impact of the Niche” by Herold-Mende and Mock [7] gradually expands our understanding of the complexity of gliomas and provides additional therapeutic targets: the interactions with stromal cells, immune cells, their adjacent endothelial cells and the extracellular matrix. Immune targeting therapies are also likely to benefit from a better understanding of glioma biology related to cancer stemness. It was recently found that the cancer stem cell subtype classified as CD133+ CSCs (proneural-like CSCs) or CD133− CSCs (mesenchymal-like CSCs) may determine the response to immunotherapy and survival [8]. The original research article “Tapasin and human leukocyte class I dysregulation correlates with survival in glioblastoma multiforme” by Thuring et al, adds additional support correlating the dysregulation of tapasin and human leukocyte class I with survival of patients with GBM [9]. The influence of HLA-2 antigen presentation and tapasin is discussed in detail in the review by Darabi et al., “HLA-I antigen presentation and tapasin influence immune responses against malignant brain tumors – considerations for successful immunotherapy”, that brings discussion on IFNγ therapy, cytostatics and irradiation [10]. Successful treatment of gliomas would likely require multimodality treatments and thus a better integrated knowledge of glioma biology is essential. The article “Translational gap in glioma research” by Ma et al. [11] raises awareness of the poor success on translating basic research into clinical trials. The potential of local delivery and nanodrugs-based therapies as way to increase the effectiveness of anticancer drugs and overcome the blood brain barriers is reviewed in the article “Overcoming the blood-brain barrier for chemotherapy: limitations, challenges and rising problems” by Wang et al. [12]. Due to the heterogeneity and complexity of glioma tumors prolonged treatment with high doses of anticancer agents will be necessary to cure cancer. Indeed few attempts has been done recently in this direction: i) a recent study showed that prolonged administration of adjuvant temozolomide improved the survival in adult patients with glioblastoma [13], ii) a two phase treatment using prolonged high exposure to anticancer drugs (e.g. alkylating agents or DNA replication inhibitors) followed by prolonged esposure to low concentration of salinomycin has been shown in vitro to prevent regrowth of glioma surviving cells [14, 15]. iii) a new proposal to increase overall survival and quality of life include adding already-marketed growth factor-inhibiting drugs to low dose continuous temozolomide [5]. Future improvements in drug delivery either by local or nanoparticles-based therapies in combination with prolonged treatments with less toxic drugs will be important to overcome the challenges due to tumor heterogeneity and the presence of the blood brain barrier. The potential tumorigenic properties of all glioma cancer cells likely due to their plasticity properties and its modulation by the microenvironment that allow interconversion between glioma stem cells and non-glioma stem cells [16-19] is a novel concept that is gradually being accepted as one of the major challenge to completely eradicate the tumor and prevent tumor relapse. The concept that to cure gliomas all cancer cells should be eliminated at once [16, 20] is now replacing the classical belief that cancer stem cells should be the main therapeutic target. For instance Schonberg et al. recently stated “To achieve a brain cancer cure, all tumor cells, particularly the brain cancer stem cells, must be eliminated” [21]. To be successful, other types of therapies not discussed in this hot topic issue but actively investigated (e.g. gene therapy, boron neutron capture therapy) should also take in consideration these novel concepts of glioma biology. At present nobody can predict which type of therapy will be the effective one and therefore any promising strategy is worth pursuing. However, as our knowledge of the complexity of gliomas increases it is likely that a multidisciplinary approach will be more advantageous over a single monotherapy modality. Novel multidisciplinary strategies need to be explored at earlier stages (during the preclinical development) and integrated during early clinical trials to avoid the translational gap of glioma research that if not corrected the prognosis of patients will remain as poor as it has been for the last five decades.

There is a lot of experimental evidence that brain tumors might be sustained by a subpopulation of immature cells, so-called brain tumor stem cells (BTSCs), which do not only drive tumor formation but are highly resistant to conventional therapies. Recent findings suggest a critical role of the molecular and cellular tumor microenvironment in which these cells reside for the maintenance of stem cell properties and therapy resistance. However, detection of different BTSC phenotypes even in the same patient tumor and the observation of a marked plasticity due to instability of the BTSC phenotype caused by the environmental niche have led to a controversial discussion on the validity of the cancer stem cell concept. What complicates the situation even more is that there are different types of niches and little is known about the interplay of the niche components with one another and with different types of BTSCs in the context of stem cell maintenance. In this article we review our current knowledge on different BTSC phenotypes and the cellular components and physiology of the niche in which these cells reside. In addition, we will summarize the molecular and functional interaction of niche cells and niche conditions and how this impacts on BTSC maintenance.

Glioblastoma Multiforme (GMB) is the most aggressive primary brain tumor with poor survival rates and universal recurrence despite aggressive treatments. Recent research suggested that GBM has multiple glioma cell populations, some of which are organized in a stem cell hierarchical order with different stages of differention. Evidence indicated that recurrence is due to a development or persistance of a subpopulation of these tumor cells which are inherently resistant to treatment and these were defined as the glioma stem-like cells (GSC). It is hypothesized that GSC become highly malignant by accumulating mutations in oncogenic pathways. These cells present with specific surface markers which helps identify them. Targeting the surface markers as well as the signaling pathways of GSCs has been an ongoing research effort. This review focuses on summarizing the current treatment modalities used to glioblastoma treatments, evaluating their efficacy in controlling and eradicatig the GSCs, discussing the machanisms involved in GSC tumor proliferation and resistance to treatments in addition to proposing potential avenues to target GSCs in order to provide a potential cure for this cancer.

Treatment of brain tumors with chemotherapy is limited mostly because of delivery impediments related to the blood-brain barrier (BBB). For gliomas, the most common and aggressive primary brain tumor, treatment includes surgery, radiotherapy, and chemotherapy usually administered orally or intravenously. These routes do not deliver effective concentrations. To complicate matters, chemotherapy is usually a long treatment. Therefore, transient disruption of the BBB is likely insufficient to deliver effective intratumoral concentrations of anticancer drugs. This review briefly updates current strategies for overcoming the BBB with emphasis on their limitations and challenges intrinsic to the biology of cancer cells.

Human leukocyte antigen class I (HLA-I) presents antigenic peptides to cytotoxic CD8+ T cells (CTLs). This is a pivotal step in the generation of CTL responses. Both the quantity and quality of peptide-HLA-I (pHLA-I) complexes are crucial for CTL responses, but the level of HLA-I expression per se is also directly involved in dictating NK-cell responses. Antigen processing machinery (APM) proteins are involved in the maturation of HLA-I and in the selection of which peptides are – or are not – presented. Thus, these proteins are key players in shaping the immune response to cells in health and disease. In this review, we recap the most important features of APM components and their synergistic work to assure proper pHLA-I cell surface expression. We pay special attention to the HLA-I dedicated multifunctional protein, tapasin, and in relation to the different tapasin-dependency of HLA-I allomorphs we also discuss allomorph specific traits in maturation, structure and linkage to malignant diseases and brain tumors in particular. We next discuss the possibilities of restoring or manipulating the immune responses against brain tumors. In this context we discuss IFNγ therapy, cytostatics and irradiation. Finally, we integrate current views and knowledge to set the direction for future emphasis in the area of immunotherapy against brain tumors.

Human leukocyte antigen class I (HLA-I) molecules present antigenic peptides to cytotoxic CD8+ T cells. Downregulation of peptide:HLA-I complexes is common in tumors and results in tumor immune escape variants. Also molecules involved in the maturation of HLA-I have been demonstrated to be dysregulated in malignant neoplasms. We here set out to investigate the antigen presentation capabilities of a set of 12 glioblastoma multiforme (GBM) tumors based on the expression of HLA-I. Moreover, we analyzed the expression of tapasin, a protein dedicated and essential to HLA-I maturation, as well as the infiltration of CD8+ cells using immunohistochemistry on paraffin-embedded sections. Comparison of different GBMs showed a variation in expression of both HLA-I heavy chain (HC) and tapasin. Interestingly, the expression of tapasin and HLA-I HC correlated significantly (p=0.0002) suggesting tapasin to be a key factor for efficient HLA-I antigen presentation in GBMs. Although no statistically significant correlation between CD8+ cells and survival was found, probably due to a very low number of infiltrating CD8+ cells at the time of surgical resection, both tapasin and HLA-I HC levels significantly correlated with survival. We suggest that analysis of expression of tapasin and/or HLA-I may be of value as prognostic tool for GBM patients, especially when considering immunotherapy.

During the last five decades, enormous advances in treatment modalities for cancer and a better understanding of cancer cell biology have been accomplished but the prognosis of patients carrying malignant gliomas still remains poor despite hundreds of clinical trials have been carried out. In this article we review phase II clinical trials that have been completed and published in PubMed during 2011 in order to investigate potential reasons of clinical failure. We suggest that a translational gap, defined as a failure to translate basic research into clinical trials design may explain the poor outcome of phase II clinical trials.

Resistance to chemotherapy, biological and targeted therapies is an important clinical problem. Resistance can arise and/or be selected for multiple mechanisms of action. Unfortunately, acquired resistance to antitumor agents or regimens is nearly inevitable in all patients with metastatic disease. Until recently, it was believed that this resistance was unalterable and irreversible, rendering retreatment with the same or similar drugs futile in most cases. However, the introduction of epigenetic therapies, including HDAC inhibitors and DNA methyltransferase inhibitors (DNMTIs), has provided oncologists with new strategies to potentially overcome this resistance. For example, if chemoresistance is the product of multiple non-genetic alterations, which develop and accumulate over time in response to treatment, then the ability to epigenetically modify the tumor to reconfigure it back to its baseline non-resistant state, holds tremendous promise for the treatment of advanced, metastatic cancer. This minireview aims (1) to explore the potential mechanisms by which a group of small molecule agents including HDACs (entinostat and vorinostat), DNA hypomethylating agents such as the DNMTIs (decitabine (DEC), 5-azacytidine (5-AZA)) and redox modulators (RRx-001) may reprogram the tumors from a refractory to non-refractory state, (2) highlight some recent findings in this area, and (3) discuss the therapeutic potential of resensitization approaches with formerly failed chemotherapies.

Cellular and molecular mechanisms related to lung cancer have been extensively studied in recent years, but the availability of effective treatments is still scarce. Hecogenin acetate, a natural saponin presenting a wide spectrum of reported pharmacological activities, has been previously evaluated for its anticancer/antiproliferative activity in some in vivo and in vitro models. Here, we investigated the effects of hecogenin acetate in a human lung cancer cell line. A549 non-small lung cancer cells were exposed to different concentrations of hecogenin acetate and reactive species production, ERK1/2 activation, matrix metalloproteinase expression, cell cycle arrest and cell senescence parameters were evaluated. Hecogenin acetate significantly inhibited increase in intracellular reactive species production induced by H2O2. In addition, hecogenin acetate blocked ERK1/2 phosphorylation and inhibited the increase in MMP-2 caused by H2O2. Treatment with hecogenin acetate induced G0/G1-phase arrest at two concentrations (75 and 100 µM, 74% and 84.3% respectively), and increased the staining of senescence-associated β -galactosidase positive cells. These data indicate that hecogenin acetate is able to exert anti-cancer effects by modulating reactive species production, inducing cell cycle arrest and senescence and also modulating ERK1/2 phosphorylation and MMP-2 production.

Baicalin, the main active ingredient in the Scutellaria baicalensis (SB), is prescribed for the treatment of various inflammatory diseases and tumors in clinics in China. In the present study, we evaluated the antitumor activity of baicalin for gallbladder carcinoma and the underlying mechanisms both in vitro and in vivo. Our results indicate that baicalin induced potent growth inhibition, cell cycle arrest, apoptosis and colony-formation inhibition in a dose-dependent manner in vitro. We observed inhibition of NF-κB nuclear translocation, up-regulation of Bax and down-regulation of Bcl-2, as well as increased caspase-3 and caspase-9 expression after baicalin treatment in vitro and in vivo, which indicates that the mitochondrial pathway was involved in baicalin-induced apoptosis. In addition, daily intraperitoneally injection of baicalin (15, 30 and 60 mg/kg) for 21 days significantly inhibited the growth of NOZ cells xenografts in nude mice, which improved the survival of baicalin-treated mice. In summary, baicalin exhibited a significant anti-tumor effect by suppressing cell proliferation, promoting apoptosis, and inducing cell cycle arrest in vitro, and by suppressing tumor growth and improving survival in vivo, which suggested that baicalin represents a novel therapeutic option for gallbladder carcinoma.

Cucurbitacin B (Cuc B) is a natural product with potent anti-cancer activities in solid tumors. We investigated the anti-cancer effect of Cuc B on K562 leukemia cells. Cuc B drastically decreased cell viability in a concentration-dependent manner. Cuc B treatment caused DNA damage, as shown by long tails in the comet assay and increased γH2AX protein expression. Immunofluorescence, Fluo3- AM, and JC-1 staining results showed that Cuc B treatment induced nuclear γH2AX foci, increased intracellular calcium ion concentration, and depolarized mitochondrial membrane potential (MMP), respectively. Cuc B induced G2/M phase arrest and apoptosis, as shown by flow cytometry, DNA fragmentation, and protein expression analyses. In addition, Cuc B dramatically increased intracellular reactive oxygen species (ROS) generation as measured by DCFH2-DA. N-acetyl-l-cysteine pretreatment significantly reversed Cuc B-induced DNA damage, increased intracellular calcium ion concentration, and reduced MMP, G2/M phase arrest, and apoptosis. Taken together, these results suggested that ROS mediated Cuc B-induced DNA damage, G2/M arrest, and apoptosis in K562 cells. This study provides novel mechanisms to better understand the underlying anti-cancer mechanisms of Cuc B.

Purpose: Platelet extracts can stimulate cell growth and contribute to tumor biology. It was recently shown that they stimulate the growth of hepatocellular carcinoma (HCC) cells and decrease apoptosis. Doxorubicin is a commonly used HCC chemotherapy that increases apoptosis. We therefore examined the effects of platelet lysates (hPL) on doxorubicin-mediated HCC cell growth inhibition and apoptosis induction. Methods: Three human HCC cell lines, PLC/PRF/5, Hep3B and HepG2 cells, were grown in culture and growth was measured by the MTT assay and apoptosis was measured using Muse Annexin V assay kit. Cells were also probed by Western blot. Results: hPL decreased doxorubicin-mediated growth inhibition and apoptosis induction in all three cell lines. When doxorubicin and hPL were added at separate time intervals, protection by hPL was also observed. WB showed that hPL caused prolonged and increased levels of phospho-JNK and phospho-p38. Furthermore, a p38 inhibitor abrogated the modulating effects of hPL on both growth and apoptosis, indicating its importance in mediating hPL actions. WBs also showed that hPL decreased doxorubicin-induced markers of apoptosis. Conclusions: hPL modulate the actions of the cancer chemotherapeutic agent, doxorubicin. Platelets are part of the complex microenvironmental milieu and their effects may contribute to a modulation of chemotherapy actions. Conversely, drugs that alter platelet levels or degranulation could potentially augment doxorubicin actions on HCC cells.

Recently we have reported potent anti-cancer actions of various steroidal Na+/K+ ATPase inhibitors in multiple cell lines. Furthermore, the most powerful compound identified in this study, the 3-[(R)-3-pyrrolidinyl]oxime derivative (3-R-POD), was highly effective in various tumor cell lines in vitro, and exhibited significant tumor growth inhibition in prostate and lung xenografts in vivo. In the present study we have addressed the molecular mechanisms implicated in the anti-cancer actions of 3-R-POD. We report here that 3-R-POD induces strong apoptotic responses in A549 lung- and in DU145 prostate- cancer cells. These effects are accompanied by significant upregulation of caspase-3 activity. Focussing on A549 cells, we further demonstrate late downregulation of BCL-2- and upregulation of c-Fos- gene transcription. In addition, the steroidal Na+/K+ ATPase inhibitor induced late de-phosphorylation of Focal Adhesion Kinase (FAK) and activation of p38 MAPK. Our findings suggest that the steroidal Na+/K+ ATPase inhibitor 3-R-POD induces apoptosis, paralleled by altered BCL-2 and c-Fos gene transcription, inhibition of the pro-survival FAK signalling, up-regulation of the pro-apoptotic p38 MAPK pathway and stimulation of caspase-3 activity.

Lupulone, a β-acid derived from hop extracts has been shown to exhibit cytotoxic activity against cancer cells. In this study we investigated the functional role of different modes of cell death that mediate anticancer effect of lupulone derivatives in prostate cancer cells. ELISA, immunoblotting and siRNA approaches were utilised to study cell death, expression of proteins of interest and their functional activities. We found that the anticancer effect of lupulone derivatives on prostate cancer cells is associated with induction of apoptosis and autophagy as determined by increases of DNA fragmentation and LC3I/ LC3II conversion respectively. Inhibition of apoptosis using a pan-caspase inhibitor resulted in increased levels of autophagy. Following screening of proteins associated with autophagy we found that Atg4β expression was increased in prostate cancer cells after treatment with lupulone. Transfection of cells with siRNA against Atg4β resulted in increased levels of apoptosis in prostate cancer cells. Treatment of prostate cancer cells with lupulone derivatives initiated two modes of cell death: apoptosis as a killing pathway and autophagy as a protection against cell death. Further studies are required to investigate the regulation of Atg4β activity in lupulone derivatives-induced negative crosstalk between apoptosis and autophagy.

The metabolism of alkylating agents is accompanied by the generation of reactive oxygen species. The aim of this study was to treat estrogen receptor-positive and estrogen receptor-negative human breast cancer cells, MCF-7 and MDA-MB-231, respectively, with cisplatin and five different berenil-platinum (II) complexes, and then to investigate the oxidative modifications of DNA, lipid and protein, and to compare them with the profile of various pro- and antiapoptotic proteins. Changes in the levels of 8-hydroxy-2’-deoxyguanosine, 4-hydroxynonenal, carbonyl groups, dityrosine, active caspases 3, 8 and 9, as well as the expression of Bcl-2, Bax, cytochrome c, and p53 were subsequently examined. Activities of superoxide dismutase, catalase and glutathione, vitamin C levels were also investigated. Cellular reactions to cisplatin and the berenil-platinum (II) derivatives were more pronounced in MCF-7 cells as compared with the MDA-MB231 cells. Furthermore, the berenil-platinum (II) derivatives were found to be more effective than cisplatin. All of the complexes reduced the activity of superoxide dismutase and catalase, and also lowered the levels of non-enzymatic antioxidants. Increased level of lipid, protein as well as DNA damage markers was also observed after berenil-platinum (II) derivatives treatment. Similarly, the increase in the levels of the proapoptotic factors, were detected in MCF-7 and MDA-MB231 cells. Incubation of examined cells with the berenil-platinum (II) complexes also led to the increase in the levels of active caspases 3, 8 and 9. In conclusion, the results of the present study demonstrated that berenil-platinum (II) complexes more efficiently mediate cellular oxidative modifications and proapoptotic metabolism, particularly in MCF-7 cells, compared to cisplatin. Pt2(isopropylamine)4berenil2 and Pt2(piperidine)4(berenil)2 notably affected the cellular metabolism of estrogen-positive breast cancer cells. Thus, these complexes may be valuable for the design of additional anticancer drugs.