Aldehyde Dehydrogenases (ALDH) are a group of enzymes involved in the removal of toxic products such as aldehydes from the body; by NAD coupled conversion to carboxylic acid. Many studies have confirmed that ALDH subtype 1 could function as a potential biomarker in both normal and cancerous stem cells.
In patients with cancers of the breast, lungs and the pancreas, increased expression of ALDH1 is associated with poor prognosis and worse overall survival. However, in certain tissues such as the liver, erythrocytes and the ovaries a naturally high level of ALDH is expressed. Therefore drug design needs to take into considerations mechanisms which not only detect an increase in expression of ALDH1 in tissues, but once detected to differentiate the cells whether the expression is naturally high or due to the emergence of cancer. Another major problem with drug design is the presence of resistance to therapy. ALDH1 has been confirmed in many studies to be resistance to treatment of alkylating agents such as cyclophosphamides.
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One marker currently available in the market which takes all of this into consideration is ALDEFLUOR. ALDEFLUOR works by distinguishing ……. cells from …… cells. The marker comes with an efflux inhibitor which allows the dye (BODIPY) to detain within viable cells and fluoresce.
What remains a challenge in research is identifying the stage of the cell cycle in which a normal stem cell becomes a cancerous stem cell? Whether the isolated cells are actually tumour initiating cells? And what the ideal model for stem cell research is? The ultimate evidence for human cancer stem cells would be the identification of a single cell selected from human cancer that regenerated the tumour in an experimental model.
Abbreviations: CSC (Cancer Stem Cells); ALDH (Aldehyde Dehydrogenase); CD (Cluster of differentiation); HPC (Hematopoietic Progenitor Cells); 4HC (4-Hydroxycyclophosphamide); CYP (Cyclophosphamide)
1.0 Introduction 3
1.1 What is Cancer? 3
1.2 Chemotherapy 3
1.3 What are Stem Cells? 3
1.4 Cancer Stem Cells 4
1.4 Biomarkers 4
2.0 Aims 6
3.0 Aldehyde Dehydrogenase 6
3.1 Mechanism of action 6
3.2 Aldehyde Dehydrogenase 1 7
3.4 Evidence against 10
4.0 Therapeutic opportunities 11
4.1 Is ALDH1 a potential for drug design? 12
5.0 ALDEFLUOR 12
5.1 Cell surface markers and niche interactions and resistance 13
5.1 Combination 15
6.0 Conclusions 16
6.1 Why do many therapies fail to eradicate Cancers? 16
6.2 Stem Cell research pros and cons 16
6.3 Critical summary of method 16
7.0 Gaps and Future 17
1.1 What is Cancer?
Cancer is a set of diseases characterised by unregulated cell growth (REF). It can lead to the invasion of surrounding tissues and spread (metastasis) to other parts of the body (REF). The cause of cancer is believed to involve interactions between genetic susceptibility and environmental toxins [1-2]. Cancer can affect people of all ages with the risk for most types increasing with age [3-6]. According to Cancer Research UK; Breast, Lung, Bowel and Prostate Cancers together account for over half of all new Cancers each year [7-8]. It is estimated that in the western world 1 in 3 people will develop cancer during their lifetime; of which 1 in 4 will die of Cancer. There is no magic bullet discovered for cancer thus treatment can vary depending on the tumour type, cancer type as well individual differences.
In terms of cancer treatment chemotherapy means treatment with cell killing (cytotoxic) drugs. Chemotherapy is a standard option for most types of cancer: it is often used in combination with other treatment modalities such as surgery and radiation. Chemotherapeutic agents work by entering the bloodstream and reaching all parts of the body. Depending on the type or how advanced the cancer is chemotherapy can be administered to cure, control, or ease cancer symptoms.
However, chemotherapy works by damaging dividing cells: which include both normal and cancerous cells explaining why chemotherapy can cause toxic side effects (e.g. bone marrow toxicity, damage to the skin, hair follicle and lining of the digestive system) [9-12]. It is important to acknowledge, not all patients undergoing chemotherapy benefit from treatment but will not be spared of the side-effects. The treatment regimen can also lead to death in a small group of patients and one need to understand these grave consequences before starting the treatment. The ability to differentiate between normal and cancerous cells is critical to be spared of unwanted side effects. This review will begin with examining the origins of cancer stem cells and looking at ways to differentiate them from normal stem cells by the use of biomarkers i. e. ALDH1.
1.3 What are Stem Cells?
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Critical to the developing organism Stem Cells (SCs) are unspecialised cells with the ability to replace or repair cells in the body [13-16]. SCs develop from the fertile egg and are most abundant in the foetus . They are characterised by their ability of self renewal via mitotic division and can differentiate into a diverse range of specialised cell types e.g. blood, brain, muscle hence can be broadly be characterised into adult SCs and embryonic SCs . Many pathways that were primarily described in cancer may also regulate normal SC development, for examples signalling pathways associated with oncogenesis such as Notch, Sonic, Oct-4 and Wnt pathway . Therefore it is important to differentiate not only the signalling pathway between a healthy and cancerous SC but also the stage or the triggering factor within the signalling pathway that causes a normal cell to become cancerous. Acknowledging the importance of SCs in human development; it is vital not to harm them and look for biological biomarkers to distinguish them from cancerous stem cells.
1.4 Cancer Stem Cells
The concept of cancer stem cells (CSC) is not new. The first idea of cancer and surviving cells from embryonic life was claimed through the works of Ernest McCulloch and James Till (1963) who used colonogenic assays to suggest that cancer is a colonel disease : This break-through earnt McCulloch and Till once of sciences highest recognition the Lasker award. Further work carried out by Hamurger and Selby with the use of SC assays claimed that only a small proportion of cancer cells (<1%) have the ability of generating colonies: a property exclusive to most SCs .
Although the CSC hypothesis has value the theoretical question remains regarding whether isolated cells are actually tumour initiating cells that function in the solid tumours of patients? It was noticed that some experimental mouse models of leukaemia however do not follow the hypothesis, suggesting that certain human cancers may not adhere this model [22-23]. Many cancers might contain subpopulations of tumour initiating cells [24-27], but some also could contain common tumeronegenic cells with little hierarchical organisation. The problem therefore is not to assume that every cancer follows the same model making the target even more difficult. Scientists are now looking at biomarkers which are exclusive property to certain SCs which could be targeted by chemotherapeutic agents.
The National Institute of Health (NIH) defined biomarkers as measurable and quantifiable characteristics that serve as indicators of pathological or pharmacological related events. According to Zhang, the criteria for ideal biomarkers includes 'stemness,' specificness of the cancer as well as specificness of tissue . Based upon this Douville et al suggested that Aldehyde Dehydrogenase subtype 1 could be a potential biomarker for malignant and cancerous SCs [29-30].
A very intriguing arises is at which stage of cell differentiation do CSCs arise? It is possible to argue that cancer can arise at any stage of cell differentiation, where it is likely that the early events are most likely to occur in normal SCs, as it is only these cells that have the ability to live long enough to accumulate several genetic changes required for cancer to develop . Evidence demonstrated by chronic lymphocytic leukaemia supported the clonal evolution of CSCs .
There is a study by Houghton et al 2007 which claims cancer can arise from circulating SCs which are more primitive than the tissue specific adult SCs . The study revealed development of stomach cancer resulted from bone marrow cells populating the stomach as a result of chronic infection of mice with Helicobacter Pylori. However the plausibility of this study is disputable as it has not yet been replicated.
The aim of this review is to gain a critical insight into the role of Aldehyde Dehydrogenase subtype 1 in SCs. Its role in both normal and cancerous SCs will be analysed in order to gain a deeper understanding of how ALDH1 a potential biomarker for identifying CSCs could be therapeutically manipulated for drug design. Potential drug designs will discussed based upon known strategies available in the market. By critically analysing current research and following it through the journey of ALDH discovery, gaps in research will be pointed out, as will suggestions of opportunities for future drug development will be discussed. Finally, a critical opinion on the overall topic will be given based upon understanding.
3.0 Aldehyde Dehydrogenase
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Aldehyde Dehydrogenases (ALDH) are a group of enzymes involved in the regulation of retinoic acid synthesis [34-36], clearance of toxic by-products of Reactive Oxygen Species (ROS) as well as the regulation of SCs [37-39]. ALDH appears to be highly expressed in premature hematopoietic cells and is developmentally expressed in early embryogenesis . The ALDH super-family is composed of 17 isoforms. ALDH1 is the predominant ALDH isoform in mammals that regulates the conversion of retinoaldehydes to retinoic acid [40-41]. Both ALDH1 and ALDH2 are found in many tissues throughout the body with the highest concentration in the liver , brain , retina [44-45] and the gastrointestinal tract [46-48]. ALDH2 is a cystolic enzyme with a homotetrameric structure and has been linked to ethanol induced cancers as well as the late onset of Alzheimer's disease . ALDH3 is a dimeric enzyme associated with severe genetic disorders and carcinogenesis e.g. Sjogren Larson Syndrome (ALDH3A1) . ALDH family 5 are members of the syccinic semi-Aldehyde family in plants whereas ALDH6 encode a methylmalonyl semi-Aldehyde Dehydrogenase and are involved in the degradation of caline to propionyl CoA [51-52]. ALDH7, ALDH10, ALDH11, ALDH12 protein families are involved in osmotic stress [51-55]. The diverse distribution of ALDHs throughout the body suggests its importance in normal function. Targeting with cytotoxic drugs needs to be specific by targeting only those isoforms predominantly elevated in certain cancers. Therefore the mechanism of action of why ALDHs are up regulated needs to be understood.
3.1 Mechanism of action
By using NMR analysis of the crystal structure of ALDH provides a basis to carry out advanced molecular modelling of the enzyme [56-57]. Aldehyde enters the active site which contains a ROSSMAN FOLD and interactions between cofactors and fold allow for the isomerisation of enzyme while keeping the active site functional . The mechanism of action involves binding of NAD+ binding to the substrate Aldehyde and forming a of a thiohemiacetal intermediate with an active site cysteine [58-59]. This is followed by Sulphur from cytosine in the active site making a nucleuphic attack on carbonyl group of Aldehyde. The intermediate then collapses to a thioester, followed by the donation of a hydride ion to nictinamide, thioester hydrolysis activated by a glutamate acting as a general base, and the release of the product acid resulting in NADH release (figure1)[60-62].
This review will only focus on ALDH subtype 1 which is seen in the presence of SCs,
Figure 1: Nucleophilic representation of the mechanism involved in ALDH generation
3.2 Aldehyde Dehydrogenase 1
Ginestier et al 2007 demonstrated that the first isoform of Aldehyde Dehydrogenase (ALDH1) is a marker of normal and malignant human mammary SCs and a predictor of poor clinical outcome [29, 63-64]. ALDH1 accounts for 95% of the human liver ALDH activity  and is the only ALDH isoform present in human erythrocytes  (fig 2).
Figure 2: only 1 band at isoelectric point 5.2 for Aldehyde Dehydrogenase, this pI is identical with the pI of human liver and cytosolic ALDH1
Ran et al 1999
Supporting the view that ALDH1 has an important role in breast is a study carried out by charafe-jauffret et al 2010 who investigated the role of ALDH1 in Inflammatory Breast Cancer (IBC): using both in vitro testing and mouse models. Sum149 cell lines were used from patients with primary breast cancer (<6months) as well as MARYX cells line which are a type of human IBC Xenograft . ALDH1 positive cancer cells were considered as ALDH1 positive tumours. The influence of ALDH1 being the sole marker of Breast SCs is somewhat doubted based upon the fact that only 5.96±2.2% of the SUM149 and 7.2±1.5% of the MARYX xenograft were positive for ALDH1. This allows for speculation that other isoform or maybe other markers of IBC which need to be investigated.
The investigation into the overlap between previously associated markers such as CD44+/CD24- was also investigated and revealed expression in 13.5% of the ALDH1 positive cells: suggesting that there was no significant correlation between CD44+/CD24- and ALDH1 expression. A thoughtful follow up of 67 months, ALDH1 expression was strongly correlated with tumour specific survival (P= 0.0337) and was the most powerful marker of tumour specific survival (p=0.0012).
One downfall of the study was that the mice had not hit puberty, since the majority of Breast cancer is associated with post puberty, the hormonal influences that may contribute to the development of the cancer may not be present. Another downfall is the fact that primary human breast tumours are not feasible because breast cancer has a well documented low xenograft rate.
Furhtermore in 2010 Nalwoga et al carried out a study on African Breast Cancer patients concluding that ALDH1 is a marker for SCs. The patients selected in the study had been diagnosed with breast cancer between 0.5 to 9 years. However the stage of Breast cancer was known in only 22 out of the 127 patients: histological staining was used to confirm the rest. The study failed or missed out the comparison of histological staining to the actual stage of cancer; (how viable the staining actually is unknown). Patients with shorter duration of symptoms were significantly (p =0.036) most likely to express ALDH1 than those with longer duration of symptoms suggesting increased expression of ALDH1 is associated with a more aggressive form of African breast cancer. A vairiety of markers were used oestrogen receptor, progesterone receptor, P-cadherins, BMI-1 and many others. However there was no consensus on how to define different molecular subtypes of breast cancer by immuno-histochemical markers and categories of overlapping existed. Therefore it can be assumed that expression of ALDH1 may be related to ethnic hereditary or diet. The study failed to explain reasoning behind the cause of elevation of ALDH1 expression in African Breast cancer patients in comparison to others.
Another study carried out by Ginestier et al 2007 found that only 19 and 30% of ALDH1 expression is associated in two different Caucasian populations. However there may have been discrepancies, although biological differences may be present .
In the vast majority of breast tumours analysed in this study the ALDH1 Positive cells represented a relatively small population considerate with the notation that cancer SCs constitute mainly of the tumour population. Remarkably only two tumours out of 481 analysed had a predominant ALDH1 Positive population . These tumours had a very aggressive clinical evaluation and may have been driven by SC population locked in self renewal undergoing little or no differentiation .
Increased expression of ALDH1 is also associated with Pancreas Cancers. However researchers have found that pancreatic CSCs may be responsible for resistance to chemotherapy. Rasheed and colleagues suggested that CD24+, CD44 expressed ALDH and had an increased potential for reproduction (characteristic of SCs) and may be expressed for pancreatic cancer metastasis. Cells that are positive to ALDH have worse overall survival and poor prognosis.
3.4 Evidence against
However there is some evidence in literature that suggests over-expression of ALDH1 may not be correlated with cancer. According to the findings of Chung et al 2009, over-expression of ALDH1 has a different function in ovarian cancer then its function in breast cancer
The study involved 442 patients, aged range between 21-89 years. High expression of ALDH1 (>20% expression) was significantly associated with early stage disease (P=0.006), and endometrioid adenocarcinoma (p<0.0001). The study found that patients with progression or recurrent disease had a significantly low level of ALDH1 expression (p=0.03).
Figure 3: Patients with tumours with >20% ALDH1 positive expression had a better disease free survival (p=0.006) than patients who had a low expression of ALDH1 positive cells.
ALDH1 has been demonstrated to be a stem cell marker in several types of malignancies[30, 68-70] Theoretically, a high proportion of cancer stem cells in the tumor should be correlated with a poor prognosis. However, depending on the cancer site, markers used to identify stem cells from one organ may or may not be useful for identifying stem cells from other organs or tumor types[71-72].
The study on ovarian cancer patients was based on a large number of patients and demonstrates that expression of ALDH1 is correlated with favorable outcome in patients with ovarian carcinoma. However, it remains to be determined whether ALDH1 is associated with a stem cell or stem-like cells in human ovarian cancer.
4.0 Therapeutic opportunities
ALDH has been studied extensively over the last 20 years with more and more research confirming it to be a prognostic marker. Drug design will present a challenge in ways that ALDH1 may be naturally elevated in certain tissues like the liver.
4.1 Is ALDH1 a potential for drug design?
There are many mechanisms that could be used to exploit the presence of ALDH1 in cancer; but differentiating healthy proliferating cells from cancerous cells is a major challenge whilst treating many cancers. One possible mechanism is attaching a fluorescent molecule to the COOH terminal, however it is important to consider that effects on the affinity and modification of the overall structure. There are two types of methods for labelling cells which are currently in practice. The first involves using radiopharmaceuticals such as 111inoxine (p8) 99M TcHMPEO (p7) or [18F] FDG (p7), and the second cells being transduced with reporter specific radio labelled (p7). Both methods can be purified first then manipulated ex vivo. However the two methods have other significant limitations such as physical decay of radio nucleotides which will affect long term fate of SCs.
Currently there is only one known product currently available in the market: ALDEFLUOR which is used to detect the presence of ALDH1 expression. The ALDEFLUOR assay detects intracellular ALDH expression in viable cells by using green fluorescent channels of a standard flow cytometer . Activity of ALDH is tested rather than cell surface phenotype (differentiating stem and progenitor cells).
ALDEFLUOR is supplied in the form of Bodipy-aminoacetaldehydediethyl acetal (BAAA-DA) (fig) which itself is not a substrate for ALDH. BAA-DA is dissolved in DMSO for 30 mins and exposed to acid (e.g. HCl) to convert it to BAA-DA which is a fluorescent substrate for ALDH1A1. BAA is uncharged and can diffuse freely across the plasma membrane of intact viable cells. Intracellular ALDH converts BAAA into Bodipy-aminoacetate, which is retained intracellularly because of its negative charge, which disallows free diffusion. One major downfall is that tumours have restricted blood flow, since ALDEFLOUR detects in blood it may not be useful in some solid tumours hence its low level of detection. However is effluxed out via the pump.
Only cells with an intact cellular membrane can retain the ALDEFLUOR, therefore only viable cells are identified. ALDEFLUOR staining does not effect cell proliferation and viability. The buffer contains an ATP binding Cassette (ABC) transport inhibitor that prevents active efflux of the ALDEFLOUR product from these cells. The inhibitor e.g. verapamil may not however prevent the efflux from other tissue types or from other species. The large number of erythrocytes present in peripheral blood, apherisis collections, bone marrow and umbilical cord blood samples can compete with stem/progenitor cells for the ALDEFLOR substrate.
5.1 Cell surface markers and niche interactions and resistance
An exclusive property of most CSC is the inherent drug resistance. Increasing evidence suggests that the cystolic enzyme ALDH1 may be involved in the mechanisms of cellular resistance to cyclophosphomide. In human hematopoietic progenitor cells (HPCs), a correlation has been made between cyclophosphomide resistance and ALDH1 Levels during hematopoietic differentiation. However a direct role of the coenzyme in including cyclophophomide resistance has not been formally shown.
5.1.2 Evidence supporting inherent drug resistance of ALDH1
Originally the role of ALDH1A1 was looked at in hematopoietic cells and now recently in lung cancer cells [74-76]. Two isoforms involved in causing resistance to therapy are ALDH3A1 and ALDH1A1. Moreb et al 2008 confirmed that down regulation of each enzyme by RNA anti-sence , or siRNA  results in sensitivity 4-hydroperoxycyclophosphomide (4-HC), an active derative of cyclophosphamide (CYP) [79-80].
Initial study carried out by Ren et al in 1998 claimed that human ALDH1 activity could be inhibited by a degradation product of 4-HC known as Acrolein. The investigation revealed that ALDH activity is inhibited by 85, 88 and 91% on days 1, 2 and 3 of administration of CYP respectively. The study aimed to find the mechanism of decrease of ALDH1 activity after CYP administration. Human liver tissue was used in the study and the formation rate of IAA from IAL was used to measure ALDH activity. A variety of inhibitors were tested, but acrolein seemed to have the greatest inhibition effect. Therefore the effect of acrolein in the formation of CEMP from 4-HC was evaluated in the human liver cystol. The mechanism of ALDH inhibiton was sought in ALDH1 prepared from human erythrocytes (blood from 5 patients receiving acrolein). Dialysis experiments were preformed to examine the reversibility of the inhibition of ALDH activity be acrolein.
The study reflects that activity of ALDH1 is inhibited by the prodrug cyclophosphamide, however the results were not significant. Although the study was preformed in duplicates, only blood from five patients was used, not indicating any history or state of cancer which may contribute to resistance.
Furthermore Quash et al 2002 claimed that competitive inhibitors of ALDH1 such as DiMATE could help restore the chemosensitivity in L1210 cells over-expressing ALDH1 . DIMATE 400µM showing 80% inhibition of baker's yeast ALDH1 in vitro. It is apparent that the inhibition of ALDH activity in L1210 also increases ATE concentration increases. With L1210 transfected with human ALDH the inhibition of ALDH1 activity was similar to control at 50 and 100µM (5-17% inhibition); whereas at 150µM inhibition was only 24% in transfected cell. Meaning inhibitors were not target sensitive. The study failed to identify these targets. 50µl ATE alone and HCPC 2.5uL had no effect on inhibition. Naturally expected HCPC to have no effect but combination has 27% response. However in comparison to the control 35% of the combination had an effect and 35% on its own of HCPC.
crocker k et al 2009
Killing these cells could inhibit their survival pathway or sensitize them to chemotherapeutic agents a major problem for scientists. Alternatively, differentiating the CSCs might prove to be successful strategies as the bulk of tumours have a limited proliferation potential. However, tumour SCs could regain renewal activity. It might therefore be therapeutically advantageous to combine agents that target CSCs with the conventional agents that reduce the bulk of tumour and agents that target the niche.
6.1 Why do many therapies fail to eradicate Cancers?
Some of the key possible reasons for failure to eradicate cancer include the inherent drug resistance of CSCs [82-83] the inefficiency of the treatment and or the genetic instability of cancer cells [84-85]. A therapy that kills 95% of cells in a tumour might be considered effective based upon tumour shrinkage however could potentially allow for survival of adequate CSCs to cause an eventual relapse. Meaning, even if CSCs are no more resistant to therapy than bulk tumour cells, they could present limitations for successful treatment. Nevertheless, understanding the molecular basis of CSC behaviour will allow new strategies, including combination therapies to counter drug resistance.
6.2 Stem Cell research pros and cons
The study of SC research is a very controversial topic in. From the view point of scientists SC research has played an important role in treating a range of medical problems including Parkinson's[86-87], Alzheimer's , heart disease , type 1 diabetes[90-91], birth defects as well as the replacement and the repair of damaged organs [93-97]. The reduced risk of transplantation means that one could possibly get a copy of own heart transplantation in the future. For most researchers the benefits for SC research has significant advantage and out-weights ethical issues of cost benefit analysis.
However, in relation to research and SCs it is very important to consider ethics [98-103]. Many would however disagree or maybe even refuse treatment if found the origin of treatment involved SCs claiming that the use of aborted tissue is inhumane. Furthermore embryonic SCs are far from being clinically viable because embryonic SCs have an amplificatory effects and may therefore accelerate the growth of tumours [104-105]. Generally SCs make up fewer than 5% of all cells in a tumour but may be the key to tumour progression
6.3 Critical summary of method
In terms of using ALDEFLUOR to identify the presence and role of ALDH1 in stem cells, there are some important drawbacks. ALDEFLOUR is not FDA approved for in vitro testing in the USA. Whilst most major tissues express some form of the ALDH, the ALDH expressed may not be able to oxidise the ALDEFLOUR substrate. Where ALDEFLOUR has been optimised for use with human cells its performance with other species has not yet been validated, i.e. results from experimental models may somewhat be challenged.
There may be background fluorescence which may be present due to excess substrate giving false positives. Alternatively ALDH1 negative tumours may contain rare ALDH1 positive cells below the level of detection by immunostaining of TMAs. The detection of an ALDH1 positive population in TMA core may be increased due to an increased self renewal activity of these tumours. Therefore only the cells that are replicate will be detected. Whereas the small percentage of cells could be missed out, increasing the likelihood tumour reoccurrence.
One major down is ALDEFLOUR staining is that it's not specific to ALDH1A1: Knockout mice models of ALDH1A1 had no effect ALDEFLOUR staining on HSCs and bone marrow cells: Suggesting the potential involvement of other isoform activity in mice HCs, despite the predominant expression of ALDH1A1.
7.0 Gaps and Future
There are limited examples of agents that are selectively toxic against cancer SCs. The proteasome inhibitor (bortezomib) kills leukaemia SCs by not normal haematopoietic SCs (REF). Also adhesion receptor CD44 is differentially expressed but CSCs and normal SC; therefore is a new target for antibody based therapy. New strategies need to take into account the role of the microenvironment that can have a critical role in modulating stem cell fate and response to therapeutic agents.
Something to think about, is it not too late in the stage of cancer before ALDH1 levels are high enough to be identified?