Electrochemotherapy: An Alternative Strategy for Improving Therapy in Drug-Resistant SOLID Tumors.

Electrochemotherapy (ECT) is one of the innovative strategies to overcome the multi drug resistance (MDR) that often occurs in cancer. Resistance to anticancer drugs results from a variety of factors, such as genetic or epigenetic changes, an up-regulated outflow of drugs, and various cellular and molecular mechanisms. This technology combines the administration of chemotherapy with the application of electrical pulses, with waveforms capable of increasing drug uptake in a non-toxic and well tolerated mechanical system. ECT is used as a first-line adjuvant therapy in veterinary oncology, where it improves the efficacy of many chemotherapeutic agents by increasing their uptake into cancer cells. The chemotherapeutic agents that have been enhanced by this technique are bleomycin, cisplatin, mitomycin C, and 5-fluorouracil. After their use, a better localized control of the neoplasm has been observed. In humans, the use of ECT was initially limited to local palliative therapy for cutaneous metastases of melanoma, but phase I/II studies are currently ongoing for several histotypes of cancer, with promising results. In this review, we described the preclinical and clinical use of ECT on drug-resistant solid tumors, such as head and neck squamous cell carcinoma, breast cancer, gynecological cancer and, finally, colorectal cancer.

Electrochemotherapy with Mitomycin C Potentiates Apoptosis Death by Inhibiting Autophagy in Squamous Carcinoma Cells.

We investigated the chemosensitizing effect of electroporation (EP), which, using electrical pulses, permeabilizes cancer cells to drugs. The study involved two human hypopharyngeal and tongue carcinoma cell lines. The surface and intracytoplasmic expression of P-gp were evaluated by flow cytometry, demonstrating that both lines were intrinsically resistant. After establishing the optimal dose of mitomycin C (MMC) to be used, in combination with EP, we showed, by both MTT assay and optical and electron scanning microscopy, the potentiating cytotoxic effect of EP with MMC compared to single treatments. Flow cytometry showed that the cytotoxicity of EP + MMC was due to the induction of apoptosis. In addition to verifying the release of cytochrome C in EP + MMC samples, we performed an expression analysis of caspase-3, caspase-9, Akt, pAkt, HMGB1, LC3I, LC3II, p62, Beclin1, and associated proteins with both apoptotic and autophagic phenomena. Our results were confirmed by two veterinary patients in whom the EP + MMC combination was used to control margins after the resection of corneal squamous carcinoma. In conclusion, we affirmed that the effect for which EP enhances MMC treatment is due to the inhibition of the autophagic process induced by the drug in favor of apoptosis.

Che-1/AATF-induced transcriptionally active chromatin promotes cell proliferation in multiple myeloma.

Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of "active chromatin" by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.

Preliminary assessment of electrochemotherapy feasibility in dogs with vesical transitional cell carcinoma.

Electroporation is a technique that increases the uptake of chemotherapeutic drugs by tumors. Electrochemotherapy (ECT) has been successfully used to treat solid tumors. Recently, novel applications have been explored in the treatment of visceral tumors. This report aimed to describe the ECT as an approach to vesical carcinoma in three dogs. The patients received ECT with bleomycin as an intravenous bolus and intra-lesional cisplatin (cases 2 and 3). The ECT was performed by electroporator (Onkodisruptor®) using a plate and/or a single pair needle array electrode. Case 1 was a 7-year-old female Pitbull dog with a history of hematuria and stranguria. The ECT was performed during cystotomy using a single pair array electrode. However, the patient developed uroabdomen two days post-ECT and died 5 days later. Case 2 was a 12-year-old female Poodle dog with hematuria, dysuria, and pollakiuria. Cystotomy and ECT were performed using plate array electrodes. Complete remission of the intra-luminal mass was observed 11 days post-ECT. However, 21 days after the procedure, an acute unilateral renal failure occurred possibly due to a neoplastic embolus into the right ureter leading to kidney hydronephrosis, and the patient was euthanized. Case 3 was a 10-year-old female Cocker dog with hematuria and pollakiuria. The patient was fully competent after ECT without clinical signs of pollakiuria and recovered from hematuria 7 days post-ECT. The bladder returned to normal status 28 days post-ECT. The ECT was not able to increase the overall survival of the patients evaluated and should be indicated carefully.

Drug repurposing for anticancer therapies. A lesson from proton pump inhibitors.

Introduction: Worldwide, the annual expenditure on anticancer drugs is grossly calculated to be in the order of US$100 billion, and is expected to escalate up to $150 billion by 2020. It is evident that the vast majority of the most recently devised anticancer drugs are unaffordable in economically developing nations, frequently resulting in subpar therapies. In this complex medical and economic scenario, the repurposing of older drugs for anticancer therapies becomes a necessity. The repurposing of antiacid drugs such as the proton pump inhibitors as antitumoral agents and chemosensitizers is probably one of the most recent and promising phenomenon in oncology.Areas covered: Important research articles and patents focusing on proton pump inhibitors as a potential class of therapeutics, published between the period of 2006-2019, have been covered. This review mainly focuses on the therapeutic applications, as direct anticancer agents as well as modifiers of the tumor microenvironment and modulator of chemoresistance.Expert opinion: PPIs have significant anticancer applications and are proving to be safe, effective and inexpensive. Here the authors review the current knowledge regarding the influence of PPIs on the efficacy and safety of cancer chemotherapeutics through the regulation of targets other than the H+/K+-ATPase.

Anticancer activity of "Trigno M", extract of Prunus spinosa drupes, against in vitro 3D and in vivo colon cancer models.

In 2018 there were over 1.8 million new cases worldwide of colorectal cancer and relapses after clinical treatments. Many studies ascribe the risk of the appearance of this cancer to the Western life style : a sedentary life, obesity, and low -fiber, high -fat diets can promote the onset of disease. Several studies have shown supplement phytochemicals to have an inhibiting effect on the growth of various cancers through the activation of apoptosis. Our goal was to prove the effectiveness of a natural compound in the combined therapy of colorectal cancer. Trigno M supplement was an optimal candidate as anticancer product for its high concentrations of phenolic acids, flavonoids and anthocyanins. Our work showed the antitumor activity of Trigno M, extract of Prunus spinosa drupes combined with the nutraceutical activator complex (NAC), in 2D, 3D and in vivo colorectal cancer models. The cellular model we used both in vitro and in vivo was the HCT116 cell line, particularly suitable for engraftment after inoculation in mice. Trigno M inhibited the growth and colony formation of HCT116 cells (35%) as compared to the chemotherapy treatment with 5-fluorouracil (80%) used in clinical therapy. The reduction of the morphological dimensions in the spheroid cells after Trigno M, was compared with 5-fluorouracil demonstrating the efficacy of the Trigno M compound also in 3D models. Flow cytometric analysis on 3D cells showed a significant increase in the apoptotic cell fraction after Trigno M treatment (44.8%) and a low level of necrotic fraction (6.7%) as compared with control cells. Trigno M and 5-fluorouracil induced the apoptosis in a comparable percentage. Monotherapy with Trigno M in severely immunodeficient mice, carrying colon rectal cancer xenografts, significantly reduced tumor growth. The histopatological analysis of the ectopic tumors showed a lower level of necrosis after Trigno M treatment compared with the control. We conclude that Trigno M is well tolerated by mice, delays colorectal cancer growth in these animals and should be weighed up for integration of the current multi-drug protocols in the treatment of colon carcinoma.

Electrochemotherapy in Veterinary Oncology: State-of-the-Art and Perspectives.

Tumor microenvironment represents a key obstacle for the effectiveness of anticancer drugs. Electrochemotherapy involves the systemic or local delivery of lipophobic drugs such as bleomycin and cisplatin, with the application of permeabilizing electric pulses having appropriate amplitude and waveforms. This greatly enhances the uptake of these drugs by an estimated factor of 700-fold for bleomycin and 4 to 8 times for cisplatin. Because of its efficacy and limited morbidity, this therapeutic option is becoming more and more available in veterinary oncology either as an adjuvant to surgery or as first line of treatment with palliative or curative purposes.

Causes, consequences, and therapy of tumors acidosis.

While cancer is commonly described as "a disease of the genes," it is also associated with massive metabolic reprogramming that is now accepted as a disease "Hallmark." This programming is complex and often involves metabolic cooperativity between cancer cells and their surrounding stroma. Indeed, there is emerging clinical evidence that interrupting a cancer's metabolic program can improve patients' outcomes. The most commonly observed and well-studied metabolic adaptation in cancers is the fermentation of glucose to lactic acid, even in the presence of oxygen, also known as "aerobic glycolysis" or the "Warburg Effect." Much has been written about the mechanisms of the Warburg effect, and this remains a topic of great debate. However, herein, we will focus on an important sequela of this metabolic program: the acidification of the tumor microenvironment. Rather than being an epiphenomenon, it is now appreciated that this acidosis is a key player in cancer somatic evolution and progression to malignancy. Adaptation to acidosis induces and selects for malignant behaviors, such as increased invasion and metastasis, chemoresistance, and inhibition of immune surveillance. However, the metabolic reprogramming that occurs during adaptation to acidosis also introduces therapeutic vulnerabilities. Thus, tumor acidosis is a relevant therapeutic target, and we describe herein four approaches to accomplish this: (1) neutralizing acid directly with buffers, (2) targeting metabolic vulnerabilities revealed by acidosis, (3) developing acid-activatable drugs and nanomedicines, and (4) inhibiting metabolic processes responsible for generating acids in the first place.

Electrochemotherapy for the treatment of an incompletely excised subcutaneous low-grade epithelioid hemangioendothelioma in a budgerigar parakeet (Melopsittacus undulatus).

Background: Cutaneous tumors are rarely described in avian and are frequently of viral origin. Solid tumors of vascular origin are seldom reported and usually result in difficult management by surgery alone. We describe the outcome of a subcutaneous low-grade epithelioid hemangioendothelioma (EHE) treated with the combination of surgery and electrochemotherapy. Case Description: A 10-year-old male budgerigar parakeet (Melopsittacus undulatus) was referred for evaluation of a 2-month non-healing exophytic mass on the left wing. The bird was bright, alert, and responsive, with a 2 × 1 cm proliferative lesion on the wing. Signs of discomfort were elicited by the clinical manipulation of the wing; no other abnormalities were detected during physical evaluation. Following hematological and imaging analysis, the parakeet was anesthetized and the mass was surgically removed. The histopathology report came back with a diagnosis of incompletely excised subcutaneous low-grade EHE. A surgical revision was not feasible due to the anatomical location and tumor extension. Adjuvant electrochemotherapy was chosen to increase the chance of tumor control. Two sessions of electrochemotherapy were performed with a 2-week interval between treatments using intralesional bleomycin followed by trains of permeabilizing electric pulses. Side effects were not observed and the parakeet was disease-free for 12 months when he died of acute renal failure. Conclusion: In lieu of the incomplete surgical excision of the tumor, electrochemotherapy resulted in good local control and cosmetic appearance and should be added to the standard oncological therapies for avian.

A Role of Tumor-Released Exosomes in Paracrine Dissemination and Metastasis.

Metastatic diffusion is thought to be a multi-step phenomenon involving the release of cells from the primary tumor and their diffusion through the body. Currently, several hypotheses have been put forward in order to explain the origin of cancer metastasis, including epithelial⁻mesenchymal transition, mutagenesis of stem cells, and a facilitating role of macrophages, involving, for example, transformation or fusion hybridization with neoplastic cells. In this paradigm, tumor-secreted extracellular vesicles (EVs), such as exosomes, play a pivotal role in cell communications, delivering a plethora of biomolecules including proteins, lipids, and nucleic acids. For their natural role in shuttling molecules, EVs have been newly considered a part of the metastatic cascade. They have a prominent role in preparing the so-called "tumor niches" in target organs. However, recent evidence has pointed out an even more interesting role of tumor EVs, consisting in their ability to induce malignant transformation in resident mesenchymal stem cells. All in all, in this review, we discuss the multiple involvements of EVs in the metastatic cascade, and how we can exploit and manipulate EVs in order to reduce the metastatic spread of malignant tumors.

Electrochemotherapy palliation of an oral squamous cell carcinoma in an African hedgehog (Atelerix albiventris).

A five-year-old female African hedgehog (Atelerix albiventris) was referred for a one month growing oral mass. The hedgehog was quiet, alert and responsive, with a 1.00 × 1.50 cm ulcerated lesion on the mandible. The patient was staged with total body radiographs, hematological and biochemical analyses and the mass was biopsied under anesthesia. The excised tissue was reported to be a squamous cell carcinoma. At this time, due to the extension of the mass, a surgical excision was not a therapeutic option and the tumor was palliated with electrochemotherapy (ECT). Monthly sessions of ECT were performed using intra-lesional bleomycin injection followed by trains of biphasic electric pulses. The treatment was well tolerated and the patient remained in partial remission for five months until tumor progression. At that time the pet was euthanized. The ECT resulted in improved local control and should be considered as a possible adjuvant treatment in exotic pets with advanced tumors.

Isolated limb perfusion electrochemotherapy for the treatment of an advanced squamous cell carcinoma of the hoof in a mare.

A twenty-year-old female saddle horse was referred for evaluation of a seven month, non-healing erosive lesion of the right hind hoof with proliferation and bleeding of the underlying soft tissues. This lesion had been twice surgically treated as a canker but rapidly recurred. Histological examination of the second excision revealed a well-differentiated squamous cell carcinoma. At presentation, the horse was mildly depressed, lame and partially non-weight-bearing on the right hind leg, which exhibited a 10 x 10 cm erosive and proliferative lesion remodeling the hoof. After completing staging procedures, the lesion was approached with surgery and intraoperative electrochemotherapy (ECT) administration of bleomycin in isolated limb perfusion. A second session of surgery and ECT was performed one month later, followed by three additional monthly sessions of ECT. During periodic recheck, the mare showed continuous improvement. One year after presentation, the mare was in complete remission and her gait markedly improved. ECT was well-tolerated and resulted in improved local control of a tumor in a challenging anatomical district.

Acridine Orange/exosomes increase the delivery and the effectiveness of Acridine Orange in human melanoma cells: A new prototype for theranostics of tumors.

Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466 nm. However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration. The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules. In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models. The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO. This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.

Ultrasound guided electrochemotherapy for the treatment of a clear cell thymoma in a cat.

A twelve-year-old male castrated domestic shorthair cat was presented for rapidly progressing respiratory distress. The cat was depressed, tachypneic and moderately responsive. Ultrasonography showed a mediastinal mass associated with a significant pleural effusion that needed tapping every five to seven days. Ultrasound guided biopsy yielded a diagnosis of clear cell thymoma upon histopathology. After complete staging procedures, the owner elected to treat the cat with electrochemotherapy (ECT) using systemic bleomycin. Two sessions of ultrasound guided ECT were performed at two week intervals with trains of biphasic electric pulses applied using needle electrodes until complete coverage of the area was achieved. The treatment was well tolerated and resulted in partial remission (PR). Additional sessions were performed on a monthly basis. The cat is still in PR after fourteen months. ECT resulted in improved local control and should be considered among the available adjuvant treatments in pets carrying visceral tumors.

Antitumor effect of combination of the inhibitors of two new oncotargets: proton pumps and reverse transcriptase.

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth.This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors.

Effect of Modified Alkaline Supplementation on Syngenic Melanoma Growth in CB57/BL Mice.

Tumor extracellular acidity is a hallmark of malignant cancers. Thus, in this study we evaluated the effects of the oral administration of a commercially available water alkalizer (Basenpulver®) (BP) on tumor growth in a syngenic melanoma mouse model. The alkalizer was administered daily by oral gavage starting one week after tumor implantation in CB57/BL mice. Tumors were calipered and their acidity measured by in vivo MRI guided 31P MRS. Furthermore, urine pH was monitored for potential metabolic alkalosis. BP administration significantly reduced melanoma growth in mice; the optimal dose in terms of tolerability and efficacy was 8 g/l (p< 0.05). The in vivo results were supported by in vitro experiments, wherein BP-treated human and murine melanoma cell cultures exhibited a dose-dependent inhibition of tumor cell growth. This investigation provides the first proof of concept that systemic buffering can improve tumor control by itself and that this approach may represent a new strategy in prevention and/or treatment of cancers.

Proton pump inhibitors induce a caspase-independent antitumor effect against human multiple myeloma.

Multiple Myeloma (MM) is the second most common hematological malignancy and is responsive to a limited number of drugs. Unfortunately, to date, despite the introduction of novel drugs, no relevant increase in survival rates has been obtained. Proton pump inhibitors (PPIs) have been shown to have significant antitumor action as single agents as well as in combination with chemotherapy. This study investigates the potential anti-tumor effectiveness of two PPIs, Lansoprazole and Omeprazole, against human MM cells. We found that Lansoprazole exerts straightforward efficacy against myeloma cells, even at suboptimal concentrations (50 µM), while Omeprazole has limited cytotoxic action. The Lansoprazole anti-MM effect was mostly mediated by a caspase-independent apoptotic-like cytotoxicity, with only a secondary anti-proliferative action. This study provides clear evidence supporting the use of Lansoprazole in the strive against MM with an efficacy proven much higher than current therapeutical approaches and without reported side effects. It is however conceivable that, consistent with the results obtained in other human tumors, Lansoprazole may well be combined with existing anti-myeloma therapies with the aim to improve the low level of efficacy of the current strategies.

Proton pump inhibitors while belonging to the same family of generic drugs show different anti-tumor effect.

CONTEXT: Tumor acidity represents a major cause of chemoresistance. Proton pump inhibitors (PPIs) can neutralize tumor acidity, sensitizing cancer cells to chemotherapy. OBJECTIVE: To compare the anti-tumor efficacy of different PPIs in vitro and in vivo. MATERIALS AND METHODS: In vitro experiments PPIs anti-tumor efficacy in terms of cell proliferation and cell death/apoptosis/necrosis evaluation were performed. In vivo PPIs efficacy experiments were carried out using melanoma xenograft model in SCID mice. RESULTS: Lansoprazole showed higher anti-tumor effect when compared to the other PPIs. The lansoprazole effect lasted even upon drug removal from the cell culture medium and it was independent from the lipophilicity of the PPIs formulation. DISCUSSION: These PPIs have shown different anti-tumoral efficacy, and the most effective at low dose was lansoprazole. CONCLUSION: The possibility to contrast tumor acidity by off-label using PPIs opens a new field of oncology investigation.

Microenvironment acidity as a major determinant of tumor chemoresistance: Proton pump inhibitors (PPIs) as a novel therapeutic approach.

Despite the major progresses in biomedical research and the development of novel therapeutics and treatment strategies, cancer is still among the dominant causes of death worldwide. One of the crucial challenges in the clinical management of cancer is primary (intrinsic) and secondary (acquired) resistance to both conventional and targeted chemotherapeutics. Multiple mechanisms have been identifiedthat underlie intrinsic and acquired chemoresistance: these include impaired drug uptake, increased drug efflux, deletion of receptors, altered drug metabolism, quantitative and qualitative alterations in drug targets, increased DNA damage repair and various mechanisms of anti-apoptosis. The fast efflux of anticancer drugs mediated by multidrug efflux pumps and the partial or complete reversibility of chemoresistance combined with the absence of genetic mutations suggests a multifactorial process. However, a growing body of recent evidence suggests that chemoresistance is often triggered by the highly acidic microenvironment of tumors. The vast majority of drugs, including conventional chemotherapeutics and more recent biological agents, are weak bases that are quickly protonated and neutralized in acidic environments, such as the extracellular microenvironment and the acidic organelles of tumor cells. It is therefore essential to develop new strategies to overcome the entrapment and neutralization of weak base drugs. One such strategy is the use of proton pump inhibitors which can enhance tumor chemosensitivity by increasing the pH of the tumor microenvironment. Recent clinical trials in animals with spontaneous tumors have indicated that patient alkalization is capable of reversing acquired chemoresistance in a large percentage of tumors that are refractory to chemotherapy. Of particular interest was the benefit of alkalization for patients undergoing metronomic regimens which are becoming more widely used in veterinary medicine. Overall, these results provide substantial new evidence that altering the acidic tumor microenvironment is an effective, well tolerated and low cost strategy for the overcoming of anticancer drug resistance.

Proton pump inhibitors for the treatment of cancer in companion animals.

The treatment of cancer presents a clinical challenge both in human and veterinary medicine. Chemotherapy protocols require the use of toxic drugs that are not always specific, do not selectively target cancerous cells thus resulting in many side effects. A recent therapeutic approach takes advantage of the altered acidity of the tumour microenvironment by using proton pump inhibitors (PPIs) to block the hydrogen transport out of the cell. The alteration of the extracellular pH kills tumour cells, reverses drug resistance, and reduces cancer metastasis. Human clinical trials have prompted to consider this as a viable and safe option for the treatment of cancer in companion animals. Preliminary animal studies suggest that the same positive outcome could be achievable. The purpose of this review is to support investigations into the use of PPIs for cancer treatment cancer in companion animals by considering the evidence available in both human and veterinary medicine.