Dimitrios T. Trafalis

Saffron as a Source of Novel Acetylcholinesterase Inhibitors: Molecular Docking and in Vitro Enzymatic Studies

Inhibitors of acetylcholine breakdown by acetylcholinesterase (AChE) constitute the main therapeutic modality for Alzheimers disease. In the search for natural products with inhibitory action on AChE, this study investigated the activity of saffron extract and its constituents by in vitro enzymatic and molecular docking studies. Saffron has been used in traditional medicine against Alzheimers disease. Saffron extract showed moderate AChE inhibitory activity (up to 30%), but IC(50) values of crocetin, dimethylcrocetin, and safranal were 96.33, 107.1, and 21.09 μM, respectively. Kinetic analysis showed mixed-type inhibition, which was verified by in silico docking studies. Safranal interacts only with the binding site of the AChE, but crocetin and dimethylcrocetin bind simultaneously to the catalytic and peripheral anionic sites. These results reinforce previous findings about the beneficial action of saffron against Alzheimers disease and may be of value for the development of novel therapeutic agents based on carotenoid-based dual binding inhibitors.

Lactandrate: a D-homo-aza-androsterone alkylator in the treatment of breast cancer.

SummaryThe sensitivity of breast neoplasms to hormonal control provides the basis of novel investigational treatments with steroidal alkylators. An androsterone D-lactam steroidal ester, the 3β-hydroxy-13α-amino-13,17-seco- 5α-androstan-17-oic-13,17-lactam, p-bis(2-chloroethyl)amino phenyl acetate (lactandrate) was synthesized and tested for antitumor activity against six human breast cancer cell lines in vitro and against two murine and one xenograft mammary tumors in vivo. A docking study on the binding interactions of lactandrate with the ligand-binding domain (LBD) of estrogen receptor-alpha (ERα) was inquired. In vitro testing of lactandrate cytostatic and cytotoxic activity was performed on T47D, MCF7, MDA-MB-231, BT-549, Hs578T, MDA-MB-435 breast adenocarcinoma human cell lines. In vivo testing was performed on two murine mammary tumors, the MXT tumor and CD8F1 adenocarcinoma, as well as on human mammary carcinoma MX-1 xenograft. Molecular modeling techniques were adopted to predict a possible location and interaction mode of the molecule into LBD. Lactandrate induced significantly high antitumor effect against all tested in vitro and in vivo models. The cell lines with positive ER expression found to be significantly more sensitive to lactandrate. Moreover, lactandrate found to be positioned inside the binding cavity with its steroidal moiety, whilst the alkylating moiety protrudes out of receptor’s pocket. Lactandrate produced important anticancer activity on breast cancer in vitro and in vivo. Some correlation between ER and lactandrate effect was demonstrated. Docking studies provide the basis for the structure-based design of improved steroidal alkylating esters for the treatment of estrogen-related cancers.

Research on the anti-tumour effect of steroid lactam alkylator (NSC-294859) in comparison with conventional chemotherapeutics in malignant melanoma.

Evidence indicating that hybrid steroid compounds of anti-cancer agents produce reduced toxicity, significantly lower than the cytotoxic components alone, and increased anti-cancer activity has prompted the design and development of such steroids, mostly alkylating esters. We investigated the in-vitro and in-vivo activity of a homo-aza-steroidal alkylating ester (HASE), in comparison with dacarbazine (DTIC), cisplatin (CPDD), carmustine (BCNU) and semustine (MeCCNU), in the treatment of malignant melanoma. Cytotoxicity was assessed in vitro by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using a panel of six human malignant melanoma cell lines, with or without the presence of rat liver microsome assay. B16 melanoma-bearing mice were used to evaluate in vivo the anti-tumour activity of the tested compounds. In all cases of in-vitro screening, HASE displayed a significantly higher (P<0.0001) cytostatic and cytotoxic effect than DTIC, BCNU and MeCCNU, but produced significantly lower (P<0.0001) activity than CPDD. HASE exhibited a significantly smaller range than CPDD between concentration levels that produced growth arrest and those that induced a cytotoxic effect against melanoma cells in vitro. The anti-tumour activity of HASE in B16 melanoma-bearing mice, as determined by tumour growth rate inhibition (<42%) and percentage survival prolongation (treated versus control, 167%), was significantly superior (P<0.001) to that achieved by DTIC, BCNU and MeCCNU and was equal to that of CPDD. HASE exhibited a toxicity similar to that of DTIC, BCNU and MeCCNU, but significantly lower than that of CPDD. It can be concluded that HASE displays significant in-vitro and in-vivo activity in the treatment of melanoma.

Preclinical studies on NSC290205 aza‐steroid alkylator activity in combination with adriamycin against lymphoid leukaemia

NSC290205 (A) is an hybrid synthetic antineoplastic ester that is a combination of a d‐lactam derivative of androsterone and an alkylating derivative of N,N‐bis(2‐chloroethyl)aniline. We tested NSC290205 for synergistic antileukaemic activity with adriamycin (ADR), (i) in vitro against the human lymphoid leukaemia cell lines: CCRF‐CEM, MOLT‐4, and RPMI‐8226, (ii) in vivo against P388 lymphocytic and L1210 lymphoid murine leukaemias (at incipient and advanced phase). Our results indicated significant cytostatic and cytotoxic synergy of NSC290205 and ADR in vitro. We further examined these results in vivo by replacing cyclophosphamide in the standard CHOP (cyclophosphamide, hydroxydaunomycin, Oncovin, prednisone) regimen with NSC290205 (AHOP) and comparing the efficiency of these two regimens in vivo. Although treatment of P388 and L1210 with cyclophosphamide or NSC290205 alone yielded equivalent results, AHOP produced a clear benefit for survival compared with CHOP against advanced leukaemias, confirming the in vitro observations [higher percentage increase in median lifespan of treated animals over the untreated (control): 188% and 239% in L1210, 308% and 353% in P388, P < 0·01, for CHOP and AHOP respectively]. AHOP also proved to be more genotoxic and cytostatic than CHOP, inducing higher sister chromatid exchange levels and cell division delays on P388 cells in vivo. NSC290205 showed superior antineoplastic potential against lymphoid leukaemia and significant synergy with ADR, producing an excellent therapeutic outcome.

Effects of homo‐aza‐steroids on acute non‐lymphocytic leukaemia cell proliferation in vitro

Summary. Homo‐aza‐steroids (modified steroid molecules) in their esterified forms have been used extensively as carrier molecules of alkylating agents against several neoplastic malignancies in vivo and in vitro. We studied the effects of two homo‐aza‐steroid carrier molecules alone, namely 3β‐hydroxy‐13α‐amino‐13,17‐seco‐5α‐androstan‐17‐oic‐13, 17‐lactam (compound 1) and 13α‐amino‐13,17‐seco‐1,3,5‐estratrien‐17‐oic‐13,17‐lactam (compound 2), on human acute non‐lymphocytic leukaemia cell proliferation in vitro.

Topotecan and pegylated liposomal doxorubicin combination as palliative treatment in patients with pretreated advanced malignant pleural mesothelioma.

BACKGROUND Malignant pleural mesothelioma (MPM) is an aggressive disease with poor or modest responses to chemotherapy and dismal prognosis. In most of the cases the scope of the treatment is only palliative. In the current study, the combination of i.v. topotecan and pegylated liposomal doxorubicin (PLD) in advanced multi-treated MPM was tested. Primary objective was palliation of the symptoms, with the secondary ones being the establishment of the regimens safety and efficacy. PATIENTS AND METHODS Nine patients were enrolled (7 males/2 females, median age 57.5 years, ECOG performance status ≤ 2), having progressed after 2 or 3 lines of chemotherapy including pemetrexed and cisplatin. Main symptoms were dyspnea, cough, chest pain, fatigue, and anorexia. The treatment included topotecan 1.2 mg/m2 i.v. on Days 1 - 3 and PLD 40 mg/m2 on Day 4, every 28 days. The patients received 4 - 8 chemotherapy cycles (median 5.8). RESULTS In all cases, symptoms were significantly improved after the 2nd treatment cycle. Respiratory function tests showed considerable enhancement, while cough and pain were drastically reduced. All patients had objective clinical benefit, 1 patient achieving partial response and 8 stable disease. Median time to progression and overall survival was 7 and 9 months, respectively. The chosen dose of the topotecan/ PLD combination was well-tolerated with no Grade 3/4 toxicities. Quality of life, as it was evaluated by the QLQ-C30 and QLQLC13 questionnaires, had improved scores especially the ones referring symptomatology. CONCLUSION The current study shows a significant palliative effect of the topotecan/ PLD combination in pretreated patients with advanced MPM.

Indications for an alternative effective treatment of head and neck squamous cell carcinoma with temsirolimus plus bevacizumab: from bench to bedside?

Head and neck squamous cell carcinoma (HNSCC) is a group of tumors known to be sensitive to chemotherapy and radiotherapy in patients who are treatment naive. However, when recurrences do occur, these tumors generally become resistant and objective responses to therapy at that point tend to be less effective. There has been an increasing interest in developing novel molecular-targeted agents that specifically modulate growth factor and signaling pathways that are unregulated in HNSCC tumor cells. Combinations of vascular endothelial growth factor and mammalian target of rapamycin inhibitors have been used in some types of neoplasms, but no such efforts have been made in HNSCC. In this study, we investigated the in vitro, in vivo, and clinical effects of the temsirolimus (mammalian target of rapamycin inhibitor, Tem) and bevacizumab (antivascular endothelial growth factor antibody, Bev) combination. In-vitro studies were carried out on the A431 human squamous epidermoid carcinoma cell line and in-vivo studies were carried out on A431 tumor cells implanted on female Nu/Nu*nuBR (athymic nude) mice. Also, the effectiveness of the Tem and Bev combination was tested clinically in two separate clinical cases of chemoresistant HNSCC. The in-vitro, in-vivo, and clinical results showed that this combination can be significantly effective. In conclusion, we discuss the theoretical basis of the molecular pharmacological interactions between Bev and Tem that could explain these good results. If the therapeutic index is ultimately well determined, the antitumor effect of Bev and Tem is very likely to yield fruitful results.

The Effect of an Estrone D-Lactam Steroid Ester Derivative on Breast Cancer Cells and Its Predicted Binding Interactions With the Ligand Binding Domain of Estrogen Receptor-α

In order to further improve the toxicity profile and the anticancer effect of chlorambucil (CBL), we have synthesized a new estrone D-lactam steroidal ester of CBL (ESBL). The aim of this study was to investigate the in vitro activity of ESBL against primary breast carcinoma (BC) cells of operable tumors in comparison with CBL. Cells derived from fresh tumor sections that were obtained from 28 postmenopausal women with ductal BC were treated with CBL and ESBL. Apoptotic cells were distinguished from viable ones with flow cytometric methods. ESBL generated a significantly higher rate of cell apoptosis and cytotoxicity than CBL. ESBL cytotoxic effect demonstrated a significant positive weak to moderate dose-dependent correlation with the ER expression. ESBL produced antineoplastic activity superior to CBL on primary BC tumors in vitro. Moreover, a docking study on the binding interactions of ESBL with the ligand binding domain (LBD) of estrogen receptor-alpha (ERalpha) was investigated. ESBL was found to be positioned inside the binding cavity with its steroidal moiety, whereas the alkylating moiety protruded out of receptors pocket.

Targeting Programmed Cell Death -1 (PD-1) and Ligand (PD-L1): A new era in cancer active immunotherapy

ABSTRACT Improved understanding of the immune system and its role in cancer development and progression has led to impressive advances in the field of cancer immunotherapy over the last decade. Whilst the field is rapidly evolving and the list of drugs receiving regulatory approval for the treatment of various cancers is fast growing, the group of PD1‐ PDL‐1 inhibitors is establishing a leading role amongst immunomodulatory agents. PD1‐ PDL‐1 inhibitors act against pathways involved in adaptive immune suppression resulting in immune checkpoint blockade. Within the last four years two PD‐1 and three PD‐L1 inhibitors have been utilized in clinical practice against a variety of malignancies. Focus was initially placed on targeting cancers considered immunogenic such as melanoma, renal and lung cancers but subsequently the application expanded to include amongst others Hodgkin Lymphoma, urothelial as well as head and neck cancer. This article provides a comprehensive review of the early and late phase trials that led to the regulatory approval of all five PD1‐ PDL‐1 inhibitors in the corresponding cancer types. It presents available data on the combinations of PD1‐ PDL‐1 inhibitors with other therapies (immunotherapy, targeted therapy and chemotherapy), the toxicity profile of the PD1‐ PDL‐1 inhibitors and ongoing trials testing the efficacy of these agents in cancer types beyond those that have been addressed already. Finally, current and future challenges in the application of PD‐1 and PD‐L1 inhibitors are discussed with emphasis on the role of predictive biomarkers.

Adriamycin in combination with dexamethasone and octreotide lacks activity on the treatment of a 4t1 metastatic breast cancer model

The aim of this study was to evaluate whether the palliative treatment for metastatic disease with dexamethasone (DEX) plus octreotide (OCT) can improve the anticancer effects of the standard treatment with adriamycin (ADR) on a 4T1 metastatic breast cancer (MBC) model. 4T1 cells were first characterized for the expression of the somatostatin receptors 1–5 and were then inoculated onto the femur of BALB/C mice. Investigation protocols used 4T1 cell proliferation and invasion assays, analysis of radiographic images of the bone metastatic lesions, and overall survival of the diseased animals. The triple combination treatment regime (ADR+OCT+DEX) was ineffective for growth inhibition and showed an antagonistic effect on ADR activity in the 4T1 cell line in both proliferation and invasion assays. ADR treatment following the administration of the DEX+OCT regimen decreased the anticancer activity of ADR both on the grading of the bone metastatic lesions and on the overall survival of diseased animals. Moreover, the palliation treatment with OCT+DEX and in combination with ADR rather caused disease progression of the metastatic disease and bone lesions in a 4T1 MBC model in vivo. These results suggest that the administration of the DEX+OCT regimen, although may preserve palliative effects, neutralizes or reverses the anticancer effects of ADR on a 4T1 MBC model in vitro and in vivo. The simultaneous use of these drugs should be considered carefully in clinical practice.