The aldehyde dehydrogenase (ALDH) superfamily is gaining momentum in regard to stem cell and cancer research. However, their regulation and expression in the cancer microenvironment is poorly understood. The aim of this work was to understand the role of selected ALDH isoforms (1A1, 1A2, 1A3, 1B1, 2, 3A1 and 7A1) in colorectal cancer (CRC) and explore the impact of hypoxia on their expression. CRC cell lines (HT29, DLD-1, SW480 and HCT116) were grown under normoxic or hypoxic conditions (0.1% O2) and HT29 and DLD-1 in spinner flasks to generate multicellular spheroids (MCS). Hypoxia was demonstrated to have an impact on the ALDH expression, which appeared cell-specific. Notably, ALDH7A1 was induced upon exposure to hypoxia in both HT29 and DLD-1 cells, shown to be expressed in the hypoxic region of the MCS variants and in 5/5 CRC xenografts (HT29, DLD-1, HCT116, SW620, and COLO205). ALDH7A1 siRNA knockdown studies in DLD-1 cells resulted in significant reduction of viable cells and significant increase in ROS levels, suggesting ALDH7A1 to possess antioxidant properties. These findings were further supported using isogenic H1299/RFP and H1299/ALDH7A1 lung cancer cell lines. ALDH7A1, however, was found not to be involved in inhibiting the pharmacological effect or causing resistance to different cytotoxic and molecularly targeted anticancer drugs. To unravel the functional role of ALDH7A1, 9 compounds obtained from a virtual screening of 24,000 compounds from the Maybridge collection of compounds were used to probe ALDH7A1 functional activity. One compound, HAN00316, was found to inhibit the antioxidant properties of ALDH7A1 and thus could be a good starting point for further chemical tool development. Although this study underpins a potential important role of ALDH7A1 in hypoxic CRC, further work is required to fully validate its potential as a biomarker and/or pharmacological target.

Petroleum Technology Development Fund (PTDF) Nigeria / The full text will be available at the end of the embargo period: 21st March 2026

The aldehyde dehydrogenase (ALDH) superfamily is gaining momentum in regard to stem cell and cancer research. However, their regulation and expression in the cancer microenvironment is poorly understood. The aim of this work was to understand the role of selected ALDH isoforms (1A1, 1A2, 1A3, 1B1, 2, 3A1 and 7A1) in colorectal cancer (CRC) and explore the impact of hypoxia on their expression. CRC cell lines (HT29, DLD-1, SW480 and HCT116) were grown under normoxic or hypoxic conditions (0.1% O2) and HT29 and DLD-1 in spinner flasks to generate multicellular spheroids (MCS). Hypoxia was demonstrated to have an impact on the ALDH expression, which appeared cell-specific. Notably, ALDH7A1 was induced upon exposure to hypoxia in both HT29 and DLD-1 cells, shown to be expressed in the hypoxic region of the MCS variants and in 5/5 CRC xenografts (HT29, DLD-1, HCT116, SW620, and COLO205). ALDH7A1 siRNA knockdown studies in DLD-1 cells resulted in significant reduction of viable cells and significant increase in ROS levels, suggesting ALDH7A1 to possess antioxidant properties. These findings were further supported using isogenic H1299/RFP and H1299/ALDH7A1 lung cancer cell lines. ALDH7A1, however, was found not to be involved in inhibiting the pharmacological effect or causing resistance to different cytotoxic and molecularly targeted anticancer drugs. To unravel the functional role of ALDH7A1, 9 compounds obtained from a virtual screening of 24,000 compounds from the Maybridge collection of compounds were used to probe ALDH7A1 functional activity. One compound, HAN00316, was found to inhibit the antioxidant properties of ALDH7A1 and thus could be a good starting point for further chemical tool development. Although this study underpins a potential important role of ALDH7A1 in hypoxic CRC, further work is required to fully validate its potential as a biomarker and/or pharmacological target. / Jordan University of Science and Technology

The full text will be available at the end of the extended embargo: 21st Feb 2026

The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-disubstituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak-to-moderately binding to DNA, and symmetrical 1,4-disubstituted compounds were shown to be non-toxic (IC50 = 100 :M) with nonsymmetrical analogues generating IC50 values in the 1-100 :M range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133⁺/ α₂β₁ hi stem cell fraction and upregulated in CD133⁻/ α₂β₁ lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells.

The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-di-substituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak*to*moderately binding to DNA, and symmetrical 1,4-di-substituted compounds were shown to be non*toxic (IC50 = 100 :/! with non-symmetrical analogues generating IC50 values in the 1-100 :/ range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133+/ α2β1hi stem cell fraction and upregulated in CD133-= α2β1lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells. / EPSRC, Biostatus / The full text was made available at the end of the re-embargo period, 1st September 2017.

Castration-resistant prostate cancer (CRPC) remains an aggressive incurable disease in men mainly due to treatment resistance. Current treatments do not effectively eradicate cancer stem cells (CSCs), which play a pivotal role in tumour maintenance, progression and drug resistance. Aldehyde dehydrogenases (ALDHs) have been used in some tumour types as CSC markers. Their high expression and high functional activity found in CSCs is also associated with drug resistance. Emerging evidence suggests deregulation of certain ALDH isoforms have implications in cancer. The role of ALDHs in prostate cancer as potential biomarkers and therapeutic targets has not been fully explored yet. Accordingly, this study investigated the expression, regulation and function of selected ALDH isoforms in prostate cancer. This study showed that ALDH1A3, ALDH1B1, ALDH2 and ALDH7A1 are highly expressed in primary prostate cancer cells (n=9) compared to benign (n=9) prostate cells. The expression of ALDH1A3 was high in the stem cells (SCs) (n=3) as well as the more differentiated counterparts (n=16). Treatment of both benign and malignant primary prostate cancer cells with all-trans retinoic acid (atRA) also resulted in increased expression of ALDH1A3 and ALDH3A1, supporting a feedback loop between atRA and ALDHs. Furthermore, SerBob, Bob and LNCaP cells were sensitive to treatment with epigenetic drugs and led to significantly higher expression of ALDH1A2, ALDH3A1 and ALDH7A1 respectively. Importantly, siRNA suppression of ALDH1A3 and ALDH7A1 led to reduced SC properties of primary prostate cultures including reduced cell viability, migration and colony formation, and increased differentiation of transit amplifying (TA) cells to committed basal (CB) cells. Novel ALDH-affinic probes showed reduced cell viability of primary prostate epithelial cultures as a single agent and also when used in combination with docetaxel. The results indicate the potential of using ALDH-affinic compounds as single agents for therapeutic intervention or in combination with docetaxel to sensitise resistant cells to this anticancer drug. The data in this thesis provides novel findings, which supports ALDH1A2, -1A3 and -7A1 as potential biomarkers and/or therapeutic targets for drug intervention. Although, a study analysing a larger number of samples is necessary to fully understand ALDH isoform expression in CSC, TA and CB cells it is envisaged that an ALDH-targeted therapy have potential in future treatment strategies for prostate cancer. / Prostate Cancer UK