Microbial-based therapy of cancer: current progress and future prospects

The use of bacteria in the regression of certain forms of cancer has been recognized for more than a century. Much effort, therefore, has been spent over the years in developing wild-type or modified bacterial strains to treat cancer. However, their use at the dose required for therapeutic efficacy has always been associated with toxicity problems and other deleterious effects. Recently, the old idea of using bacteria in the treatment of cancer has attracted considerable interest and new genetically engineered attenuated strains as well as microbial compounds that might have specific anticancer activity without side effects are being evaluated for their ability to act as new anticancer agents. This involves the use of attenuated bacterial strains and expressing foreign genes that encode the ability to convert non-toxic prodrugs to cytotoxic drugs. Novel strategies also include the use of bacterial products such as proteins, enzymes, immunotoxins and secondary metabolites, which specifically target cancer cells and cause tumor regression through growth inhibition, cell cycle arrest or apoptosis induction. In this review we describe the current knowledge and discuss the future directions regarding the use of bacteria or their products, in cancer therapy.     

Statins,stem cells,and cancer

The statins (3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors) were proven to be effective antilipid agents against cardiovascular disease. Recent reports demonstrate an anticancer effect induced by the statins through inhibition of cell proliferation, induction of apoptosis, or inhibition of angiogenesis. These effects are due to suppression of the mevalonate pathway leading to depletion of various downstream products that play an essential role in cell cycle progression, cell signaling, and membrane integrity. Recent evidence suggests a shared genomic fingerprint between embryonic stem cells, cancer cells, and cancer stem cells. Activation targets of NANOG, OCT4, SOX2, and c‐MYC are more frequently overexpressed in certain tumors. In the absence of bona fide cancer stem cell lines, human embryonic stem cells, which have similar properties to cancer and cancer stem cells, have been an excellent model throwing light on the anticancer affects of various putative anticancer agents. It was shown that key cellular functions in karyotypically abnormal colorectal and ovarian cancer cells and human embryonic stem cells are inhibited by the statins and this is mediated via a suppression of this stemness pathway. The strategy for treatment of cancers may thus be the targeting of a putative cancer stem cell within the tumor with specific agents such as the statins with or without chemotherapy. The statins may thus play a dual prophylactic role as a lipid‐lowering drug for the prevention of heart disease and as an anticancer agent to prevent certain cancers. This review examines the relationship between the statins, stem cells, and certain cancers. J. Cell. Biochem. 106: 975–983, 2009. © 2009 Wiley‐Liss, Inc.     

Quiescence and attenuated DNA damage response promote survival of esophageal cancer stem cells

Accumulating evidence indicates cancer stem cells (CSCs) possess the capability to resist DNA‐damage induced cell death, whereas the mechanism is largely unknown. Here we show that cell cycle status and DNA damage response (DDR) in CSCs probably contribute to their survival in genotoxic insults. In this study, we isolated esophageal cancer stem cells (ECSCs) from esophageal cancer cell line EC9706 by side‐population (SP) phenotype through flow cytometry and found that ECSCs preferentially stay quiescent as compared to the non‐ECSCs and are more resistant to DNA damage agents. Further study revealed that ECSCs express a lower level of EGFR, phosphoralated Stat3, and c‐Myc, yet abnormally upregulated p27. More interestingly, different from non‐ECSCs, when suffering DNA damage agents, ECSCs showed attenuated DDR, as well as declined DNA repair potential. These data indicated ECSCs probably employed an impaired DDR to handle severe genomic insults. Conclusively, we infer that the damage‐resistance ability of ECSCs is likely attributed to their slow‐cycling status and avoidance of apoptosis or senescence triggered by an excessive DDR. J. Cell. Biochem. 113: 3643–3652, 2012. © 2012 Wiley Periodicals, Inc.     

Sorafenib combined vitamin K induces apoptosis in human pancreatic cancer cell lines through RAF/MEK/ERK and c‐Jun NH2‐terminal kinase pathways

Apoptosis has been shown to be induced by many agents, including the clinically useful Sorafenib and K vitamins (VKs). Since few agents have activity against pancreas cancer cell growth, we evaluated the role of naturally occurring K vitamins and Sorafenib both independently and together on the growth in culture of pancreas adenocarcinoma cell lines, including PL‐5, PANC‐1, and MIA PaCa‐2. We found that when a K vitamin was combined with Sorafenib, the dose of Sorafenib required for growth inhibition was substantially reduced. Furthermore, growth could be inhibited at doses of each VK plus Sorafenib in combination that were ineffective when used alone. This effect was seen using vitamins K1, K2, and K5. The combination of VK1 plus Sorafenib‐induced apoptosis, as determined by both FACS and TUNEL staining. Phospho‐ERK and Bcl‐2 levels were decreased, but not levels of other bcl‐2 family members. Cleavage of caspases 3 and 8, PARP and Bid were all induced by this combination. Vitamin K1 plus Sorafenib combination also resulted in elevated levels of activated c‐Jun N‐terminal kinase (JNK) and its substrates c‐Jun and FasL. JNK inhibition partly antagonized the induction of apoptosis. Thus, combination VK1 plus Sorafenib strongly induced growth inhibition and apoptosis in pancreas cancer cells, involving both inhibition of the RAF/MEK/ERK pathway as well as activation of the JNK, c‐Jun and FasL apoptotic pathway. Since both agents are available for human use, the combination is attractive for evaluation against pancreas cancer growth in vivo. J. Cell. Physiol. 224:112–119, 2010 © 2010 Wiley‐Liss, Inc.     

Engineering of bacterial strains and their products for cancer therapy

The use of live bacteria in cancer therapies offers exciting possibilities. Nowadays, an increasing number of genetically engineered bacteria are emerging in the field, with applications both in therapy and diagnosis. In parallel, purified bacterial products are also gaining relevance as new classes of bioactive products to treat and prevent cancer growth and metastasis. In the first part of the article, we review the latest findings regarding the use of live bacteria and products as anti-cancer agents, paying special attention to immunotoxins, proteins, and peptides. In particular, we focus on the recent results of using azurin or its derived peptide as anticancer therapeutic agents. In the second part, we discuss the challenges of using metagenomic techniques as a distinctive approach for discovering new anti-cancer agents from bacterial origin.     

Mitochondria as therapeutic targets for cancer stem cells

Cancer stem cells(CSCs) are maintained by theirsomatic stem cells and are responsible for tumor initiation, chemoresistance, and metastasis. Evidence for the CSCs existence has been reported for a number of human cancers. The CSC mitochondria have been shown recently to be an important target for cancer treatment, but clinical significance of CSCs and their mitochondria properties remain unclear. Mitochondriatargeted agents are considerably more effective compared to other agents in triggering apoptosis of CSCs, as well as general cancer cells, via mitochondrial dysfunction. Mitochondrial metabolism is altered in cancer cells because of their reliance on glycolytic intermediates, which are normally destined for oxidative phosphorylation. Therefore, inhibiting cancer-specific modifications in mitochondrial metabolism, increasing reactive oxygen species production, or stimulating mitochondrial permeabilization transition could be promising new therapeutic strategies to activate cell death in CSCs as well, as in general cancer cells. This review analyzed mitochondrial function and its potential as a therapeutic target to induce cell death in CSCs. Furthermore, combined treatment with mitochondriatargeted drugs will be a promising strategy for the treatment of relapsed and refractory cancer.     

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S‐phase arrest and apoptosis

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27‐mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27‐overexpressing cells displayed an early S‐phase arrest subsequently followed by a strongly increased sub‐G1 fraction. Apoptosis was characterized by PARP‐, CASPASE 3‐, CASPASE 8‐, CASPASE 9‐ and BIM‐ activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock‐induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27‐overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor‐targeting agents, suggesting another pro‐apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well‐established anti‐apoptotic properties of HSP27 in cancer, our study reveals novel pro‐apoptotic functions of HSP27—mediated through both the intrinsic and the extrinsic apoptotic pathways—at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients.     

Biological control of microbial infections and cancer in humans: Historical use to future potential

Prior to the advent of antibiotics, live organisms were used directly in attempts to control microbial infections and cure cancers. Examples of such biological control included bacteriotherapy, bacteriophage therapy, malaria therapy, probiotics and the use of living maggots. In all cases, the organisms themselves, rather than products of their metabolism, were used as the potentially curative agents. The history of the use of biocontrol agents in the treatment of human infections and cancer is discussed here in relation to more recent examples of the use of this approach. Modern studies suggest that the use of biological control in the treatment of human infections may be worth re‐evaluating in the light of the increasing world‐wide occurrence of antibiotic‐resistant bacteria and the opportunities provided by recent developments in gene technology.     

Control of cell proliferation in cancer prevention.

Control of cell proliferation is important for cancer prevention since cell proliferation has essential roles in carcinogenesis including the process of initiation and promotion. In rodent models for carcinogenesis, especially those for the carcinogenesis in digestive organs such as colon, liver or oral cavity, chemopreventive agents suppress carcinogen-induced hyperproliferation of cells in the target organs during the initiation as well as the postinitiation phases. Therefore, effective agents usually suppress cell proliferation and inhibit the occurrence of malignant lesions. Availability of new biomarkers for cell proliferation, apoptosis or telomerase activity could be promising. By combining the use of intermediate biomarkers including premalignant lesions such as aberrant crypt foci in the colon or enzyme-altered foci in the liver and cell proliferation, short-term screening of effective chemopreventive agents will be possible.     

Cell‐bound exopolysaccharide from probiotic bacteria induces autophagic cell death of tumour cells

Aim: Lactic acid bacteria (LAB) are beneficial micro‐organisms that have been associated with several probiotic effects in both humans and animals. Here, using proteome analysis, we investigate the antitumour effects of cell‐bound exopolysaccharides (cb‐EPS) isolated from Lactobacillus acidophilus 606 on colon cancer cells and explore the proteins critical for their antitumour activity. Methods and Results: cb‐EPS inhibited the proliferation of HT‐29 colon cancer cells by directly affecting cell morphology and not the cell cycle. Using two‐dimensional polyacrylamide gel electrophoresis coupled with matrix‐assisted laser desorption‐ionization time‐of‐flight mass spectrometry (MALDI‐TOF/MS) and immunoblot analysis, we found that cb‐EPS dramatically induced Beclin‐1 and GRP78, and affected Bcl‐2 and Bak regulation. Conclusions: The results of this study indicate that cb‐EPS are antitumourigenic against HT‐29 colon cancer cells and that this activity is because of the activation of autophagic cell death promoted directly by the induction of Beclin‐1 and GRP78, as well as indirectly through the induction of Bcl‐2 and Bak. Significance and Impact of the Study: These results may contribute to understanding the novel mechanisms by which probiotic bacteria induce tumour cell death via autophagy.     

Enucleation: a possible mechanism of cancer cell death

There are few major morphologies of cell death that have been described so far: apoptosis (type I), cell death associated with autophagy (type II), necrosis (type III) and anchorage‐dependent mechanisms—anoikis. Here, we show for the first time a possibly novel mechanism inducing tumour cell death under in vitro conditions—enucleation. We pursued the influence of colloidal suspensions of Fe₃O₄ nanoparticles on tumour cell lines (SK‐BR‐3 and MCF‐7 breast cancer cell lines) grown according to standard cell culture protocols. Magnetite nanoparticles were prepared by combustion synthesis and double layer coated with oleic acid. Scanning and transmission electron microscopy revealed that tumour cells developed a network of intracytoplasmic stress fibres, which induce extrusion of nuclei, and enucleated cells die. Normal adult mesenchymal stem cells, used as control, did not exhibit the same behaviour. Intact nuclei were found in culture supernatant of tumour cells, and were visualized by immunofluorescence. Enucleation as a potential mechanism of tumour cell death might open new horizons in cancer biology research and development of therapeutic agents capable of exploiting this behaviour.     

Identification of single chain antibodies to breast cancer stem cells using phage display

Recent evidence suggests that most malignancies are driven by “cancer stem cells” sharing the signature characteristics of adult stem cells: the ability to self renew and to differentiate. Furthermore these cells are thought to be quiescent, infrequently dividing cells with a natural resistance to chemotherapeutic agents. These studies theorize that therapies, which effectively treat the majority of tumor cells but ‘miss’ the stem cell population, will fail, while therapies directed at stern cells can potentially eradicate tumors. In breast cancer, researchers have isolated ‘breast cancer stem cells’ capable of recreating the tumor in vivo and in vitro. Generated new tumors contained both additional numbers of cancer stem cells and diverse mixed populations of cells present in the initial tumor, supporting the intriguing self‐renewal and differentiation characteristics. In the present study, an antibody phage library has been used to search for phage displayed‐single chain antibodies (scFv) with selective affinity to specific targets on breast cancer stem cells. We demonstrate evidence of two clones binding specifically to a cancer stem cell population isolated from the SUMl59 breast cancer cell line. These clones had selective affinity for cancer stem cells and they were able to select cancer stem cells among a large population of non‐stem cancer cells in paraffin‐embedded sections. The applicability of these clones to paraffin sections and frozen tissue specimens made them good candidates to be used as diagnostic and prognostic markers in breast cancer patient samples taking into consideration the cancer stern cell concept in tumor biology. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Nucleases in homologous recombination as targets for cancer therapy

Genomic DNA is constantly challenged from endogenous as well as exogenous sources. The DNA damage response (DDR) mechanism has evolved to combat these challenges and ensure genomic integrity. In this review, we will focus on repair of DNA double-strand breaks (DSB) by homologous recombination and the role of several nucleases and other recombination factors as suitable targets for cancer therapy. Their inactivation as well as overexpression have been shown to sensitize cancer cells by increasing toxicity to DNA-damaging agents and radiation or to be responsible for resistance of cancer cells. These factors can also be used in targeted cancer therapy by taking advantage of specific genetic abnormalities of cancer cells that are not present in normal cells and that result in cancer cell lethality.     

Engineering bacteria toward tumor targeting for cancer treatment: current state and perspectives

One of the primary limitations of cancer therapy is lack of selectivity of therapeutic agents to tumor cells. Current efforts are focused on discovering and developing anticancer agents that selectively target only tumor cells and spare normal cells to improve the therapeutic index. The use of preferentially replicating bacteria as an oncolytic agent is one of the innovative approaches for the treatment of cancer. This is based on the observation that some obligate or facultative anaerobic bacteria are capable of multiplying selectively in tumors and inhibiting their growth. Meanwhile, bacteria have been demonstrated to colonize and destroy tumor, and have emerged as biological gene vectors to tumor microenvironment. To improve the efficacy and safety of the bacterial therapy, a further understanding of bacteria between with immune system is required. Furthermore, we want to evaluate how bacterial infection facilitates the “bystander effect” of chemotherapeutic agent and assess if it can be used for additional antitumor effect when combined with chemotherapy. This study may not only evaluate therapeutic efficacy of bacteria for the treatment of cancer but also elucidate the mechanisms underlying antitumor activities mediated by bacteria, which involve host immune responses and the cellular molecular responses.     

Urokinase-targeted recombinant bacterial protein toxins-a rationally designed and engineered anticancer agent for cancer therapy

Urokinase-targeted recombinant bacterial protein toxins are a sort of rationally designed and engineered anticancer recombinant fusion proteins representing a novel class of agents for cancer therapy.Bacterial protein toxins have long been known as the primary virulence factor(s) for a variety of pathogenic bacteria and are the most powerful human poisons.On the other hand,it has been well documented that urokinase-type plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR),making up the uPA system,are overexpressed in a variety of human tumors and tumor cell lines.The expression of uPA system is highly correlated with tumor invasion and metastasis.To exploit these characteristics in the design of tumor cell-selective cytotoxins,two prominent bacterial protein toxins,i.e.,the diphtheria toxin and anthrax toxin are deliberately engineered through placing a sequence targeted specifically by the uPA system to form anticancer recombinant fusion proteins.These uPA system-targeted bacterial protein toxins are activated selectively on the surface of uPA systemexpressing tumor cells,thereby killing these cells.This article provides a review on the latest progress in the exploitation of these recombinant fusion proteins as potent tumoricidal agents.It is perceptible that the strategies for cancer therapy are being innovated by this novel therapeutic approach.     

The impact of type 2 diabetes and antidiabetic drugs on cancer cell growth

Despite investigations into mechanisms linking type 2 diabetes and cancer, there is a gap in knowledge about pharmacotherapy for diabetes in cancer patients. Epidemiological studies have shown that diabetic cancer patients on different antidiabetic treatments have different survival. The clinically relevant question is whether some antidiabetic pharmacotherapeutic agents promote cancer whereas others inhibit cancer progression. We investigated the hypothesis that various antidiabetic drugs had differential direct impact on cancer cells using four human cell lines (pancreatic cancer: MiaPaCa2, Panc‐1; breast cancer: MCF7, HER18). We found that insulin and glucose promoted cancer cell proliferation and contributed to chemoresistance. Metformin and rosiglitazone suppressed cancer cell growth and induced apoptosis. Both drugs affected signalling in the protein kinases B (AKT)/mammalian target of rapamycin pathway; metformin activated adenosine monophosphate (AMP)‐activated protein kinase whereas rosiglitazone increased chromosome ten level. Although high insulin and glucose concentrations promoted chemoresistance, the combination of metformin or rosiglitazone with gemcitabine or doxorubicin, resulted in an additional decrease in live cancer cells and increase in apoptosis. In contrast, exenatide did not have direct effect on cancer cells. In conclusion, different types of antidiabetic pharmacotherapy had a differential direct impact on cancer cells. This study provides experimental evidence to support further investigation of metformin and rosiglitazone as first‐line therapies for type 2 diabetes in cancer patients.     

Cancer heterogeneity—a multifaceted view

Cancers of various organs have been categorized into distinct subtypes after increasingly sophisticated taxonomies. Additionally, within a seemingly homogeneous subclass, individual cancers contain diverse tumour cell populations that vary in important cancer‐specific traits such as clonogenicity and invasive potential. Differences that exist between and within a given tumour type have hampered significantly both the proper selection of patients that might benefit from therapy, as well as the development of new targeted agents. In this review, we discuss the differences associated with organ‐specific cancer subtypes and the factors that contribute to intra‐tumour heterogeneity. It is of utmost importance to understand the biological causes that distinguish tumours as well as distinct tumour cell populations within malignancies, as these will ultimately point the way to more rational anti‐cancer treatments.EMBO reports advance online publication 12 July 2013; doi:10.1038/embor.2013.92     

Sirtuins at the crossroads of stemness,aging, and cancer

Sirtuins are stress‐responsive proteins that direct various post‐translational modifications (PTMs) and as a result, are considered to be master regulators of several cellular processes. They are known to both extend lifespan and regulate spontaneous tumor development. As both aging and cancer are associated with altered stem cell function, the possibility that the involvement of sirtuins in these events is mediated by their roles in stem cells is worthy of investigation. Research to date suggests that the individual sirtuin family members can differentially regulate embryonic, hematopoietic as well as other adult stem cells in a tissue‐ and cell type‐specific context. Sirtuin‐driven regulation of both cell differentiation and signaling pathways previously involved in stem cell maintenance has been described where downstream effectors involved determine the biological outcome. Similarly, diverse roles have been reported in cancer stem cells (CSCs), depending on the tissue of origin. This review highlights the current knowledge which places sirtuins at the intersection of stem cells, aging, and cancer. By outlining the plethora of stem cell‐related roles for individual sirtuins in various contexts, our purpose was to provide an indication of their significance in relation to cancer and aging, as well as to generate a clearer picture of their therapeutic potential. Finally, we propose future directions which will contribute to the better understanding of sirtuins, thereby further unraveling the full repertoire of sirtuin functions in both normal stem cells and CSCs.     

Comparison of patient‐derived high and low phosphatidylserine‐exposing colorectal carcinoma cells in their interaction with anti‐cancer peptides

Current cancer treatment is frequently compromised by severe adverse effects on healthy cells and tissues as well as by the increasing burden of (multi‐)drug resistances. Some representatives of small, amphipathic peptides known as host defense peptides possess the potential to overcome these limitations and to evolve as future anti‐cancer therapeutics. Peptide NK‐2, derived from porcine NK‐lysin, was originally discovered due to its broad‐spectrum antimicrobial activities. Today, also potent anti‐cancer activity is proven and accompanied by low toxicity towards normal human cells. The molecular basis underlying this target selectivity remains rather elusive. Nevertheless, it is presumptive that preferential peptide interactions with surface factors non‐abundant on healthy human cells play a key role. Here, we investigated the cytotoxicity of peptide NK‐2 and structurally improved anti‐cancer variants thereof against two patient‐derived colorectal cancer cell lines, exposing high and low levels of phosphatidylserine on their cell surfaces, respectively. Concluding from a range of in vitro tests involving cellular as well as lipid vesicle‐based methods, it is proposed that the magnitude of the accessible membrane surface charge is not a primarily decisive factor for selective peptide interactions. Instead, it is suggested that the level of membrane surface‐exposed phosphatidylserine is of crucial importance for the activity of peptide NK‐2 and enhanced variants thereof in terms of their cancer cell selectivity, the overall efficacy, as well as the underlying mode of action and kinetics. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.