Over the years, labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes with cytotoxic activities have been identified across various families in the plant kingdom including the Zingiberaceae. The present review discusses the distribution of these labdane-type diterpenes within the Zingiberaceae; their extraction, isolation, and characterization from the respective Zingiberaceae species; the structural similarities and differences within each group and between the different groups of the labdane-type diterpenes; and their cytotoxic activities against breast, cervical, liver, colorectal, pancreatic, lung and prostate cancer cell lines. The review will also provide insight into how the cytotoxic activities of the labdane-type diterpenes are influenced by their structural features. Cancer is a group of diseases characterized by uncontrollable cell growth. The World Health Organization (WHO) estimated in 2019 that cancer was the number one or number two cause of death among populations under the age of 70 in countries of all income levels ,4,5,6.The causes of cancer can be grouped into two categories, namely internal factors and external factors. Internal factors include genetic mutations, weakened immune system and abnormal hormone levels, while external factors include smoking, alcohol abuse, diet, sedentary lifestyle, lack of physical exercise, lack of social contact, chronic stress, pollutants, and radiation ,11,12,13Different types of treatment are available depending on the type of cancer and how advanced it is. Surgery, chemotherapy, radiation therapy, and proton therapy are conventional treatment strategies. Photodynamic therapy, photothermal therapy, gene therapy, nanoparticle-drug therapy, targeted therapy, and immunotherapy on the other hand are methods which have emerged as recently developed treatment strategies. A disadvantage of the currently available treatments, such as surgery, is the pain it causes and the potential damage to nearby tissues. Certain chemotherapy drugs can cause unintentional damage to other otherwise healthy organs. Parts of the body most commonly affected by chemotherapy drugs include bone marrow, hair follicles, and the reproductive system. There are also risks of rejection or infection from stem-cell or bone marrow transplant, and the whole process might take a long time. In addition, the costs of these treatments can be prohibitive, especially for patients from developing countries ,15.The currently available treatment strategies for cancer are not perfect, and there continues to be a need for more research to be carried out in order to increase the efficacy and reduce the side effects of cancer treatment. Natural anticancer agents, especially those isolated from the plant kingdom, can be a great alternative to otherwise more expensive drugs used for treating cancer. Approximately, 60% of the drugs in use today in cancer therapy were first obtained from various natural sources, and of those, many were sourced from varying species in the Kingdom Plantae .Catharanthus roseus (L.) G. Don, that has been used effectively in treating various cancers such as leukemia, lymphoma, breast cancer, and more. Vinblastine, another anticancer agent extracted alongside vincristine from the same source, is also effective in battling similar types of cancers as vincristine [Taxus brevifolia Nutt., and it is used extensively in the treatment of a variety of cancers, including ovarian cancer, breast cancer, and lung cancer [The plant kingdom is a rich source of phytochemicals which cover a wide and diverse range of products, among which include alkaloids, terpenoids, flavonoids, and other types of polyphenolic compounds. These plant-derived products possess a broad spectrum of pharmacological activities which enable them to play important roles in the prevention or treatment of many diseases, including cancer ,16. Vinccristine . These vcristine . Anotherg cancer . This tag cancer .The Zingiberaceae, commonly known as the ginger family, is one of the largest families of the plant kingdom . It is aPhytochemical investigation of the Zingiberaceae has led to the isolation and characterization of various classes of secondary metabolites such as essential oils, flavonoids , terpenoids , diarylalkanoids or arylalkanoids , phenylpropanoids  and phenolics . These secondary metabolites exhibit numerous pharmacological activities, namely anticancer, antioxidant, antimicrobial, antiviral, anti-inflammatory, anti-allergic analgesic, hypoglycemic, neuroprotective, cardiovascular-protective, and digestive-system-protective activities ,27,28,29The current paper reviews the published scientific literature related to the cytotoxic activities of the secondary metabolites isolated from the Zingiberaceae, with the emphasis being on the labdane-type diterpenes, a sub-class of the diterpenoids. This review aims to provide insight into the cytotoxic potential of the labdane-type diterpenes isolated and characterized from the Zingiberaceae that may have the potential to be developed into safe and affordable chemotherapy drugs. This review will act as a database and a foundation for further research on cytotoxic labdane-type diterpenes, which were not only isolated from the Zingiberaceae but also other families in the plant kingdom.This systematic review was carried out according to the guideline requirements of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The authors K.J.V. and A.L. independently performed the data extraction and data synthesis for this systematic review using the PICO framework . Studies applicable to the aim of this review were searched using two electronic databases: Web of Science—Core Collection and SciFinder. “Labdane diterpenes” and “cytotoxic” were the two keywords which were used to search the electronic databases for relevant articles. The complete list of keywords which were used in the search is presented in 50 and GI50 values of the in vitro cytotoxic activities were reported in this review. Studies which compared the cytotoxic activities of the naturally occurring labdane-type diterpenes with a comparator or a standard drug, or a positive control were included. Scientific research papers related to human trials, clinical trials, randomized control trials, animal studies, literature reviews, systematic reviews, meta-analyses, conference proceedings, and patents were excluded from this review. In addition, studies that did not fully report on the results or methodologies and studies in which the full text was not accessible were not included in this review.Articles were selected based on the labdane-type diterpenes isolated and characterized from the Zingiberaceae that exhibited cytotoxic activity. Only scientific research articles, including short communications, notes, and letters, written in English and published between the year 2000 and 28 March 2022 that documented the IChttps://COVIDence.org/) accessed on 14 April 2022. Cases of disagreement were resolved by another independent reviewer (Y.S.) to prevent biases.Based on the inclusion and exclusion criteria presented in A total of 997 articles were found from the search conducted using the two electronic databases. The articles were transferred into EndNote, following which the duplicates were removed and 406 articles remained. The references to these articles were then uploaded to COVIDence, where an additional 23 duplicates were removed, leaving 383 articles. The articles were first screened using their title and abstract in COVIDence, during which 291 articles were excluded, and 92 articles were left. The 92 articles were then further screened through full-text screening, where 75 articles were excluded. The reasons for the exclusion of certain articles are depicted in The chemical structures of the cytotoxic labdane-type diterpenes and their names in relation to the corresponding Zingiberaceae species are illustrated in Alpinia , Amomum (A. maximum Roxb), Curcuma , Hedychium , and Roscoea (R. purpurea Sm.)  . Among t Curcuma .The labdane-type diterpenes were extracted from either the rhizomes, roots, leaves, or the aerial parts of the different Zingiberaceae species using the solvent extraction method. The extraction of the plant materials was carried out either at room temperature or under reflux using organic solvents or aqueous alcohol .The resulting crude solvent extracts were either used directly or further partitioned with organic solvents and subjected to various chromatographic techniques, mainly silica gel column chromatography, preparative TLC, and preparative HPLC, to isolate and purify the labdane-type diterpenes .The structures of these labdane-type diterpenes were subsequently elucidated by a combination of various spectroscopic techniques such as infrared spectroscopy (IR), ultraviolet-visible spectroscopy (UV-Vis), mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) .3–27, 29–37, 39–44), two as norlabdane diterpenes  and two as bis-labdanic diterpenes   . The folAll of the labdane diterpenes in the present study consist of twenty to twenty-four carbon atoms. Their basic framework is constructed from two substructures: a decalin moiety (C-1-C-10) which consists of two fused cyclohexane rings and a branched six-membered carbon side chain  which is connected to position C-9 of the decalin moiety . The thr3–17, 20, 21, 24–27, 29–36, 43, 44) have an exomethylene group at their position C-8. As for the remaining labdane diterpenes, either a methyl , aldehyde  or hydroxymethyl  group occupied their C-8 position instead , a γ-lactone  an α,β-unsaturated γ-lactone , an epoxide (7), an unsaturated oxane (15), or an unsaturated 1,2 dioxepane (25) ring. As for compounds 5, 8, 9, 26, and 31, none of their side chains have a ring; instead, they have a combination of one of the following functional groups: carboxylic acid, aldehyde, hydroxymethyl, hydroxyl and ether  or a four-membered (28) aliphatic chain, in turn resulting in these compounds having fewer than 20 carbon atoms in their respective structures -CH3) while that of compound 38 is a propenoic acid chain (-CH=CH-COOH). In compound 28, its C-17 group is an exomethylene group, while in compound 38, it is a methyl group instead  which were identified in A. calcarata through this study are a pair of stereoisomers. Their structures are identical to one another with the exception of the orientation of the H-9 and H-9′ atoms at the corresponding C-9 and C-9′ positions of their respective decalin moieties. It is interesting to note that compounds 1 and 2 are non-symmetrical bis-labdanic diterpenes as each compound is constructed from two different monomers. Both monomers in each compound are linked together via an ether linkage  and two known labdane diterpenes (ottensinin and 5) from the dichloromethane soluble fraction of the aqueous ethanolic extract of the roots of A. maximum. When this group of researchers examined the cytotoxicity of these labdane diterpenes against the MCF-7 cell line using the MTT assay, they found compound 4 to be two times more effective (IC50 = 29.80 µM) compared to 5-fluorouracil (IC50 = 59.96 µM), the synthetic positive control which was used in their study [In 2014, Luo and his coworkers isolated and characterized three new labdane diterpenes , bisdemethoxycurcumin (IC50 = 33.78 µM), and curcumin (IC50 = 10.44 µM) were also isolated alongside compound 6 and assessed for their potential in inhibiting the cytotoxic activity of the same cell line. These compounds were not as cytotoxic as compound 6 . A new labdane diterpene glucoside, curcumanggoside, along with compounds 26 and 31, was also identified in the rhizomes of C. mangga. However, these labdane-type diterpenes were not evaluated for their cytotoxicity against the MCF-7 cell line. A separate study conducted ten years later reported a weaker cytotoxicity (IC50 = 39.71 µM) of compound 6 which was obtained from a different source: the rhizomes of H. ellipticum [50 values of compound 6 between both studies could be attributed to the differences in the assay procedures performed by the respective group of researchers. Abas et al. investigated the cytotoxicity of compound 6 using the MTT assay, while Songsri and Nuntawong employed the REMA technique instead [50 values in both of the studies [According to an investigation carried out by Abas et al. back in 2005, compound  0.59 µM . Demethoy IC50 = .71 µM ofn IC50 = .78 µM, ay IC50 = .71 µM of studies .H. ellipticum, in a phytochemical investigation carried out by Songsri and Nuntawong, yielded ten compounds in total: eight labdane diterpenes  and two norlabdane diterpenes (28 and (E)-14,15,16-Trinorlabda-8(17),11-dien-13-oic acid). According to the results obtained from the REMA assay, only five of these compounds  were cytotoxic against the MCF-7 cell line. Except for compound 16 (IC50 = 8.75 µM) which had an IC50 value comparable with that of doxorubicin (IC50 = 1.22 µM), the cytotoxicity of the rest of the compounds was considered to be either moderate (29) or weak  [20 and 31 upon comparison to compounds 6, 16, and 29 implied that the presence of an α,β-unsaturated γ-lactone ring in the side chain played an essential role in mediating the cytotoxic activity against the tested cell line. When the cytotoxicity of compounds 16 and 29 was compared to compound 6, the greater activity of compounds 16 and 29 led to the assumption that the potency of both these compounds could have resulted from the presence of a trans double bond which is conjugated to the α,β-unsaturated γ-lactone ring in their respective side chains, in contrast to compound 6 in which the trans double bond is absent. The variation in the α,β-unsaturated γ-lactone rings of compounds 16 and 29 could further explain the significant difference in the cytotoxicity between both of these labdane diterpenes. The carbonyl group being at position C-15 of the α,β-unsaturated γ-lactone ring in compound 16, instead of position C-16 as in compound 29, in addition to the presence of a hydroxyl group at position C-16 of the α,β-unsaturated γ-lactone ring in compound 16 and the absence of it in compound 29, could have increased the cytotoxicity of compound 16 to three folds of that of compound 29.The hexane and dichloromethane extracts of the rhizomes of  20, 31) . The ratH. spicatum exhibited a rather mild cytotoxic activity (IC50 = 58.08 µg/mL) against the MCF-7 cell line [40 and 41) along with the known labdane diterpene (32), although reported to be weakly cytotoxic against the MCF-7 cell line, could have been responsible for the extract’s cytotoxicity. Reddy et al. concluded that upon comparing the activity of compound 40 with that of compounds 32 and 41, the presence of an α,β-unsaturated γ-lactone ring in the side chain along with the presence of a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 of the decalin moiety of compound 40 enhanced its cytotoxicity against the tested cell line. They further deduced that even with the presence of a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 of the decalin moiety, the cytotoxicity in compound 41 was decreased due to the absence of the α,β-unsaturated γ-lactone ring. The remaining labdane diterpenes, hedychilactone B and hedychilactone C—which were also identified in the chloroform extract—were found to be inactive [In an investigation by Reddy and his coworkers, the chloroform extract which they had prepared from the rhizomes of ell line . Phytochinactive .R. purpurea and assessed its cytotoxicity against the MCF-7 cell line. The results obtained from their MTT assay revealed that the chloroform fraction was moderately cytotoxic (IC50 = 46.64 µg/mL) and slightly stronger in its activity compared to the methanol extract (IC50 = 48.96 µg/mL) itself. Purification of the chloroform fraction led to the isolation and characterization of three weakly cytotoxic labdane diterpenes, 14, 43, and 44, with the latter two compounds being identified for the first time in the plant kingdom [A year ago, Singamaneni et al. prepared a chloroform fraction from the methanol extract of the rhizomes of c IC50 = .64 µg/mL10, 18–24 which they had isolated and characterized from the hexane extract of the rhizomes of H. coronarium against the MCF-7 cell line. The results revealed that the order of their growth-inhibitory activity was 19 > 22 > 23 > 20 > 10 > 21 > 24 > 18 .In 2010, Suresh et al. employed the SRB assay to compare the growth-inhibitory properties of compounds H. coronarium. Their investigation led to the identified two positional isomers (17 and 20) differing in the position of the hydroxyl and carbonyl groups in the γ-lactone-rings of their respective labdane framework. A new labdane diterpene, 7β-hydroxy-(E)-labda-8(17),12-diene-15,16-dial, along with known compounds , were also isolated and characterized alongside the positional isomers. Chimnoi and his coworkers subsequently determined the cytotoxicities of both the positional isomers against the MDA-MB-231 cell line through the MTT assay. Compounds 17 and 20 were both considered to be equally strong inhibitors (IC50 < 8 µM) of the cytotoxic activity of the MDA-MB-231 cell line [In 2009, Chimnoi et al. reinvestigated the chemical constituents of the dichloromethane extract of the rhizomes of ell line .20 reported less than half of that activity against the T-47D cell line, while compound 17 reported almost equal cytotoxicity against the T-47D cell line, albeit to a slightly lesser degree [50 values of compounds 17 and 20 against both the MDA-MB-231 and T-47D cell lines could be attributed to the fact that the two cell lines belong to different subtypes of breast cancer: basal-type and luminal-type, respectively. These subtypes of breast cancer are associated with different gene expressions and, generally, patients afflicted with basal-type breast cancers have a worse prognosis when compared to patients suffering from luminal-type breast cancers [Compared to the strong cytotoxic activity against the MDA-MB-231 cell line, compound r degree . The dif cancers .C. mutablis for their cytotoxicity against the MDA-MB-231 cell line [7) which was found to be responsible for the activity of the petroleum ether extract. Compound 7 (IC50 = 7.71 µM) was strongly cytotoxic and four-fold more potent compared to that of the positive control, curcumin (IC50 = 31.22 µM), a diarylheptanoid [Recently, in 2021, Soumya et al. used the MTT assay to evaluate the various solvent extracts  of the rhizomes of ell line . All of eptanoid ,37.50 values between the cell lines.The cervical cancer cell lines KB, SMMC-7721, SGC-7901, and HeLa were used to perform the cytotoxic assays in the studies included in this review. KB, SMMC-772,1 and SGC-7901 were originally attributed as oral, liver, and gastric cancer cell lines in the studies obtained through our search. However, these cell lines have recently been proven to have been misidentified and are actually derivatives of the HeLa cell line ,51,52,531 and 2) from the ethyl acetate fraction of the methanol extract of the rhizomes of A. calcarata. Based on the results obtained from their MTT assay, both compounds 1 (IC50 = 0.34 µM) and 2 (IC50 = 0.24 µM) can be regarded as potent inhibitors of the cytotoxic activity of the KB cell lines due to their IC50 values being significantly comparable to the positive control, adriamycin (IC50 = 0.15 µM) [In 2002, Kong et al., isolated and characterized two novel stereoisomeric bis-labdanic diterpenes (0.15 µM) .H. ellipticum against the KB cell line. Only six of them were active. For compound 16 (IC50 = 2.75 µM), although its cytotoxic activity was strong and almost comparable to ellipticine (IC50 = 1.23 µM), it was found to be slightly weaker than that of doxorubicin (IC50 = 0.22 µM) [6, 27, 29, 30, and 31, these compounds can be regarded as being either moderate (29) or weak  [16 and 29, which could have given rise to their potency against the MCF-7 cell line as discussed in Section MCF-7 Cell Line, may also be responsible for their potency against the KB cell line. Although compound 30 is almost identical in structure to compound 29, the presence of a methoxyl group at position C-15 of its α,β-unsaturated γ-lactone ring could have considerably suppressed its activity by five-fold in comparison to compound 29.In addition to the MCF-7 cell line, Songsri and Nuntawong also assessed the cytotoxic potentials of the ten labdane-type diterpenes which they had identified in the hexane and dichloromethane extracts of the rhizomes of 0.22 µM) . As for  30, 31) . The sam17 and 20) isolated and characterized from the rhizomes of H. coronarium against the KB cell line through the MTT assay. Their findings revealed that both compounds exhibited strong cytotoxic activity  against the KB cell line with similar inhibiting potentials [Chimnoi et al. also determined the cytotoxicities of the two positional isomers  from the roots of A. maximum was once again identified by Lou and his coworkers as a more effective inhibitor (three folds) compared to the synthetic positive control, 5-fluorouracil (IC50 = 77.64 µM). The cytotoxicity of the remaining labdane diterpenes identified in this plant was considered to be inactive against this cell line [With regard to the cytotoxic activity of the SMMC-7721 cell line, compound ell line .42) along with four known labdane diterpenes  from the ethyl acetate fraction of the ethanol extract of the rhizomes of H. yunnanense. According to the results from their SRB assay, compounds 22 and 29 exhibited moderate cytotoxic activities against the SGC-7901 cell line with similar inhibiting strengths. Compound 42, on the other hand, was a weaker inhibitor. The remaining labdane diterpenes were considered inactive [22 and 42 possessed identical decalin moieties, with each compound having a double bond between positions C-7 and C-8, a carbonyl group at position C-6 and a methyl group at position C-8, the variation in their respective side chains could have attributed to the differences in their potency against the SGC-7901 cell line. The presence of a furan ring in compound 22 could have increased its activity by almost two folds compared to compound 42 which instead has an α,β-unsaturated γ-lactone ring bearing a methoxyl group at position C-15.Li and his coworkers in 2016 isolated and characterized two new labdane diterpenes (hedychenoid A and inactive . Althoug36) together with three known ones  from the rhizomes of H. longipetalum [32, 35, and 36 were moderately cytotoxic, with the former two exhibiting similar cytotoxic strength [34 and 35 being structurally similar to one another, the presence of the carbonyl group at position C-15 along with the hydroxyl group at position C-16 of the α,β-unsaturated γ-lactone ring in compound 35 may have increased its potency against the SGC-7901 cell line by two-fold in comparison to compound 34, whose carbonyl group is at position C-16 and lacks a hydroxyl group in its α,β-unsaturated γ-lactone ring.In a separate study, Zhao et al. isolated and characterized three new labdane diterpenes -labda-8(17),12-diene-15,16-dial, 17, 20 and 25 from the rhizomes of H. coronarium against the HeLa cell line. They reported that compounds 17 (IC50 = 9.12 µM) and 25 (IC50 = 8.54 µM) were strong inhibitors with almost similar ability in inhibiting the cytotoxic activity of the HeLa cell line. Compound 20, however, was only a moderate inhibitor while the new labdane diterpene, 7β-hydroxy-(E)-labda-8(17),12-diene-15,16-dial, was inactive [Chimnoi et al. in their investigation also evaluated the cytotoxicities of 7β-hydroxy-, with compounds 8 and 9 being identified as new. Only six of these labdane diterpenes were found to be cytotoxic, two of them moderately (15 and 16) and four of them weakly  [10 and 13 even though both compounds were almost structurally identical to each other. The additional presence of a methoxyl group at position C-15 in the γ-lactone ring of compound 10 could have suppressed its cytotoxicity by two and a half folds compared to compound 13. Instead, in compound 16, when a trans double bond is conjugated to an α,β-unsaturated γ-lactone ring bearing a hydroxyl group at position C-16, this structural feature could have notably increased its cytotoxicity by almost four-fold compared to compound 10.The preliminary screening of the ethyl acetate fraction of the aqueous ethanolic extract of the rhizomes of ell line . Subsequ 13, 14) . A signiH. longipetalum against the HeLa cell line [32, 34–36). Compounds 32 and 35 were found to moderately inhibit the cytotoxic activity of the HeLa cell lines, while the remaining two compounds were less effective inhibitors [Similarly, the SRB method was used to compare the cytotoxic potentials of the six labdane diterpenes from the rhizomes of ell line . In thishibitors . The samH. coronarium against the HeLa cell line. The results from their SRB assay showed that only compounds 18–24 weakly inhibited the growth of the HeLa cell line, while the remaining labdane diterpenes were found to be inactive (GI50 > 100 µM) [Suresh and his coworkers in 2010 also assessed the growth-inhibitory properties of the labdane diterpenes, which they had identified in the rhizomes of  100 µM) .Cytotoxicity assays of compounds performed on HepG2 and HCC-S102 liver cancer cell lines are reported in this review .A. intermedia were initially extracted with 95% ethanol, following which the aqueous ethanolic extract was partitioned between hexane and water. When the aqueous ethanolic extract and the hexane fraction were evaluated for their cytotoxicity against the HepG2 cell line using the MTT assay, the hexane fraction (IC50 = 66.50 µg/mL) was found to be more cytotoxic compared to the aqueous ethanolic extract (IC50 > 100 µg/mL). The phytochemical investigation of the hexane fraction yielded two new labdane diterpenes (3 and intermedin B) and a known labdane diterpene (27). Intermedin B is a C-12 epimer of 3, and both these labdane diterpenes were structurally related to 27 . When Chen and his coworkers assessed the cytotoxic potential of compounds 3 and 27 towards the HepG2 cell line, they discovered that compound 3 (IC50 = 119.82 µM) exhibited a cytotoxic activity almost identical to that of the natural anticancer compound, andrographolide (IC50 = 111.94 µM), a labdane diterpene previously isolated and characterized from Andrographis paniculata (Burm. fil.) Nees. As for compound 27, its cytotoxicity was almost three-fold weaker compared to compound 3 and andrographolide [In an investigation conducted by Chen et al. in 2017, the leaves of apholide .5 from the roots of A. maximum for its cytotoxicity towards the HepG2 cell line, the MTT assay results revealed that although compound 5 had only an IC50 value of 30.1 µM, it possessed stronger cytotoxic activity against the cancer cell when compared to the synthetic positive control, 5-fluorouracil, which had an IC50 value of more than double (IC50 = 76.11 µM) that of compound 5 [When Lou et al. evaluated compound mpound 5 .6 from the rhizomes of C. manga against the HepG2 cell line, and it was regarded as moderately cytotoxic compared to its ability in inhibiting the activity of the MCF-7 cell line. Demethoxycurcumin (IC50 > 50 µM), bisdemethoxycurcumin (IC50 > 50 µM), and curcumin (IC50 = 33.53 µM) which were also identified in the rhizomes were only considered to be weakly cytotoxic against the HepG2 cell line [Abas et al. also investigated the cytotoxicity of compound n IC50 = .53 µM whH. coronarium prepared by Zhan and his coworkers was also found to be cytotoxic against the HepG2 cell line in addition to the HeLa cell line [8–17) which were isolated and characterized from the active extract, were investigated for their cytotoxicity. Taxol, a tetracyclic diterpenoid, was used as the positive control [15, 16, and 17 were highly effective (IC50 < 10 µM) in inhibiting the cytotoxic activity of the HepG2 cells, with the former two compounds being almost equally potent inhibitors  and relatively comparable to taxol (IC50 = 0.9 µM). The remaining compounds exhibited moderate cytotoxic activities. Compounds 13 (IC50 = 10.6 µM) and 14 (IC50 = 10.7 µM) possessed almost identical cytotoxicity, while the cytotoxic activity of compounds 8, 9, 10, 11, and 12 increased in the following order: 12 > 11 > 10 > 8 > 9 [10, 11, and 13 each possessed a γ-lactone ring in their respective side chains, it is evident that the presence of either a methoxyl or ethoxyl group at position C-15 of the γ-lactone ring in compounds 10 and 11 caused a slight decrease in their respective activities compared to the activity of compound 13 in which those oxygenated functional groups are absent from position C-15 of its γ-lactone ring. Upon further comparison of the activity of compound 13 with that of compound 17, although they are structurally similar to one another, the presence of a hydroxyl group at position C-14 of the γ-lactone ring in compound 17 slightly increased its activity compared to compound 13 in which the hydroxyl group is absent from position C-14 of its γ-lactone ring. The variation in the α,β-unsaturated γ-lactone rings of compounds 12 and 16 could explain the significant difference in the cytotoxicities between both of these labdane diterpenes. The carbonyl group being at position C-15 instead of position C-16, in addition to the presence of a hydroxyl group at position C-16 of the α,β-unsaturated γ-lactone ring in compound 16, could have increased its cytotoxicity to almost twelve folds compared to that of compound 12 whose carbonyl group is at position C-16 instead of position C-15 and lacks a hydroxyl group in its α,β-unsaturated γ-lactone ring. It is noteworthy to mention that the unsaturated oxane ring in compound 15 could have attributed to its remarkable potency when compared to the potencies of the other compounds in which their side chains bear either a furan ring, a γ-lactone ring, an α,β-unsaturated γ-lactone ring or oxygenated functional groups.The ethyl acetate fraction of the aqueous ethanolic extract of the rhizomes of ell line . The ten control . Compoun > 8 > 9 . Althoug17 according to an earlier study conducted by Chimnoi and his coworkers, who also isolated this labdane diterpene from the same plant, the rhizomes of H. coronarium [17 was a moderate inhibitor with only half the cytotoxic strength [50 values between both of the studies could be attributed to the different types of assays which were used to determine the cytotoxicity of the compound. Zhan and his coworkers investigated the cytotoxicity of compound 17 using the SRB assay, while Chimnoi and his team of researchers employed the MTT method instead. Chimnoi et al. also evaluated the cytotoxic potentials of 7β-hydroxy-(E)-labda-8(17),12-diene-15,16-dial, 20, and 25. Compound 20, unlike its positional isomer, 17, showed a weak cytotoxicity against the HepG2 cell line, while compound 25 and 7β-hydroxy-(E)-labda-8(17),12-diene-15,16-dial were moderately cytotoxic and inactive, respectively [There was a difference in the cytotoxic inhibiting potential for compound ronarium . In contstrength ,37. The 17 and 20 against the HCC-S102 cell line. Interestingly, although compound 17 was moderately cytotoxic towards the HepG2 cell line, yet it was only weakly cytotoxic towards the HCC-S102 cell line with almost twice the IC50 value with that recorded against the HepG2 cell line [50 values and resulted in its susceptibility to the cytotoxic activity of compound 17. Compound 20 once again was weakly cytotoxic [In addition to the HepG2 cell line, Chimnoi et al. further assessed the cytotoxicity of compounds ell line . HepG2 iell line ,57. The ytotoxic .Cytotoxic assays against the following colorectal cancer cell lines HCT-116, HT-29, COLO 205, and DLD-1 are included in this review .There were only two recent studies which evaluated the cytotoxicity of the labdane-type diterpenes from the Zingiberaceae against this cell line.7 to be strongly cytotoxic. Compound 7 was isolated from the petroleum ether extract of the rhizomes of C. mutablis which demonstrated a slightly stronger cytotoxicity (IC50 = 5.4 µg/mL) against the HCT-116 cell line compared to the chloroform (IC50 = 5.5 µg/mL) and acetone (IC50 = 6.0 µg/mL) extracts and a far more potent cytotoxicity compared to the methanol (IC50 = 79.5 µg/mL) extract. The IC50 value of compound 7 was reported to be 3.21 µM, which was about seven times lower when compared to the positive control curcumin (IC50 = 21.45 µM) [7 exerted its cytotoxicity towards the cancer cells by the induction of apoptosis and the suppression of genes related to cell survival and migration, while also being safe for normal cells at the same concentration [7 was identified to be 2.5-fold more effective in inhibiting the cytotoxic activity of the HCT-116 cell line compared to the MDA-MB-231 cell line [The first study by Soumya et al. in 2021 revealed compound 1.45 µM) . Compounntration . Based oell line .14, 43, and 44) from the chloroform fraction of the methanol extract of the rhizomes R. purpurea against the HCT-116 cell line. Compound 43 was found to moderately inhibit the cytotoxic activity of the cells while compounds 14 and 44 were only weakly cytotoxic [14 and 43 both have a hydroxyl group at position C-7 of their respective decalin moieties and a side chain bearing a furan ring. However, in compound 14, the presence of a trans double bond conjugated to the furan ring and the absence of it in compound 43 could be a reason for the two folds decrease or more in the cytotoxic activity of compound 14 against the HCT-116 cell line when compared to that of compound 43.In another study conducted the same year, Singamaneni et al. investigated the cytotoxic potentials of the labdane diterpenes  and 11 (IC50 = 9.8 µM). Compounds 8, 9, and 13–16 on the other hand were moderately cytotoxic with relatively similar inhibitory strengths [10, 11, and 13 each possessed a γ-lactone ring in their respective side chains, it is evident that the presence of either a methoxyl or ethoxyl group at position C-15 of the γ-lactone ring in compounds 10 and 11 resulted in an increase in their respective activities compared to the activity of compound 13 whose γ-lactone ring does not bear either one of those oxygenated functional groups. Upon further comparison of the activity of compound 13 with that of compound 17, although they are structurally similar to one another, the presence of a hydroxyl group at position C-14 of the γ-lactone ring in compound 17 increased its activity compared to compound 13 whose γ-lactone ring does not bear a hydroxyl group at its position C-14. The variation in the α,β-unsaturated γ-lactone rings of compounds 12 and 16 could explain the difference in the cytotoxicities between both of these labdane diterpenes. The carbonyl group being at position C-15 instead of position C-16 in addition to the presence of a hydroxyl group at position C-16 of the α,β-unsaturated γ-lactone ring in compound 16 could have decreased its cytotoxicity compared to that of compound 12 whose carbonyl group is at position C-16 instead of position C-15 and lacks a hydroxyl group in its α,β-unsaturated γ-lactone ring.In 2012, Zhan and his group of researchers examined the cytotoxic potentials of ten compounds, ll lines . Compountrengths . In geneH. spicatum to be slightly more cytotoxic against the COLO 205 cell line (IC50 = 43.14 µg/mL) compared to the MCF-7 cell line (IC50 = 58.08 µg/mL). Once again, it would be reasonable to postulate that compounds 32, 40, and 41 could have contributed to the cytotoxic activity of the chloroform extract against the COLO 205 cell line like they did with regard to the MCF-7 cell line [32, 40, and 41 which could have given rise to their potency against the MCF-7 cell line as discussed in Section MCF-7 Cell Line may also be responsible for their potency against the COLO 205 cell line [Reddy et al. in their investigation identified the chloroform extract which they had prepared from the rhizomes of ell line . The samell line .H. coronarium. The hexane fraction of its methanol extract yielded hedychiumin, a new norlabdane diterpene, two known labdane diterpenes (14 and 26), and a known norlabdane diterpene (28). Among these four compounds, only compounds 14 and 26 exhibited cytotoxic activity against the DLD-1 cell line [14, its cytotoxic potential towards the DLD-1 cell line, according to the study conducted by Chen et al., was found to be almost three times weaker compared to its cytotoxic potential against the HT-29 cell line from an earlier study by Zhan et al. [50 values.In 2017, Chen et al. conducted a phytochemical investigation of the aerial part of ell line . The remn et al. ,38. Bothn et al. ,59,60. T14, 43, and 44 which were isolated and characterized from the chloroform fraction of the methanol extract of the rhizomes of R. purpurea were recently tested for their ability in inhibiting the cytotoxic activity of the BxPC-3 cell line, and according to Singamaneni et al. who conducted the assay, only compound 14 was found to be moderately cytotoxic [14 and 44, it would be reasonable to conclude that the furan ring in the side chain of compound 14 could have promoted its cytotoxic activity compared to the α,β-unsaturated γ-lactone ring in the side chain of compound 44. Although compounds 14 and 43 are structurally similar to one another, the greater activity of compound 14 in comparison to compound 43 could be due to the presence of the trans double bond which is conjugated to the furan ring in compound 14, and the absence of it in compound 43. Singamaneni and his coworkers identified compound 14 to be more effective in inhibiting the cytotoxic activity of the BxPC-3 cell line compared to those of MCF-7 and HCT-116 [A limited number of pancreatic cancer cell lines were used to study the cytotoxic activities of the labdane-type diterpenes isolated and characterized from the Zingiberaceae, with BxPC-3 being the only type of cell line to date . Among aytotoxic . Based o HCT-116 .The studies included in this review tested the cytotoxicities of the compounds against lung cancer cell lines, namely the A-549 and NCI-H187 lung cancer cell lines .50 = 21.35 µg/mL) of the methanol extract of the rhizomes of R. purpurea was reported to be slightly more cytotoxic against the A-549 cell line compared to the methanol extract (IC50 = 25.71 µg/mL) itself [43 which exhibited moderate cytotoxicity could have mainly been responsible for the cytotoxic potential of the chloroform fraction [43 and 44 revealed that the presence of a furan ring in the side chain would be preferred for cytotoxic activity against the A549 cell line rather than the presence of an α,β-unsaturated γ-lactone ring conjugated with a trans double bond. Similarly, when the structures of compounds 14 and 43 were compared, a trans double bond conjugated to the furan ring in the side chain was found to weaken the activity of compound 14 by 4.5-folds compared to the activity of compound 43.The chloroform fraction (IC) itself . Accordifraction . A compa17, 20, and 25 from the rhizomes of H. coronarium demonstrated cytotoxicity against the A-549 cell line while the new labdane diterpene, 7β-hydroxy-(E)-labda-8(17),12-diene-15,16-dial, was inactive. Although compound 17 was only moderate in activity, it was found to be almost five times stronger in inhibiting the cytotoxic activity of the A-549 cell line compared to compounds 20 and 25 which exhibited similar inhibiting strengths one another [In another investigation by Chimnoi et al., their findings enabled us to conclude that compounds  another .H. spicatum which they had originally investigated a year earlier [v/v) instead of using chloroform as they did in their earlier phytochemical investigation. They evaluated the cytotoxic potential of the extract against the A-549 cell line using the MTT assay and found it to be mildly cytotoxic (IC50 = 63.21 µg/mL). Upon further investigation of the active extract, the purification of it followed by spectroscopic characterization of the secondary metabolites obtained from it led to the identification of a new labdane diterpene (37) a new norlabdane diterpene (38), and two known labdane diterpenes (27 and 39). All four compounds, however, were only weakly cytotoxic with compound 37 being the most cytotoxic among the four [27 and 37 have a trans double bond which is conjugated to a furan ring in their respective side chains, the presence of a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 of the decalin moiety of compound 37 could have contributed to its greater cytotoxicity against the tested cell line compared to compound 27 in which there is no double bond between positions C-7 and C-8 and no carbonyl group at position C-6 of its decalin moiety. Even with the presence of a double bond between positions C-7 and C-8 and a carbonyl group at position C-6 in the respective decalin moieties of compounds 37 and 39, the cytotoxicity of compound 39 was weaker than that of compound 37 due to the absence of a hydroxyl group at position C-7 in the decalin moiety of compound 39. The variation in the type of labdane-type diterpenes which were identified between the previous and present studies could have been due to the following factors: collection site (different country or town), altitude, climate , season, soil pH, plant maturity, genotype, variety, harvesting time, postharvest handling conditions and storage, method of extraction and isolation and purification techniques [In 2009, Reddy et al. reinvestigated the chemical constituents of the rhizomes of  earlier . In the the four . It woulchniques ,62,63,64H. forrestii yielded two new (hedyforrestin D and 15-ethoxy-hedyforrestin D) and three known  labdane diterpenes [32 and 33 were reported to demonstrate the ability to inhibit the cytotoxic activity of this cell line. Among these two labdane diterpenes, the cytotoxicity of compound 33 was found to be potent (IC50 = 0.92 µM) and comparable to the cytotoxicity of the positive control, cisplatin (IC50 = 0.77 µM) [32 may have decreased its potency by twelve-fold compared to compound 33 which bears an α,β-unsaturated γ-lactone ring in its side chain. As for hedyforrestin D (IC50 > 300.81 µM), 15-ethoxy-hedyforrestin D (IC50 > 277.40 µM), and yunnancoronarin C (IC50 > 300.81 µM), they were considered to be inactive [The phytochemical investigation of the ethyl acetate fraction of the aqueous ethanolic extract of the rhizomes of terpenes . Upon ev0.77 µM) . Althoug10, 18–24) from the hexane extract of the rhizomes of H. coronarium against the A-549 cell line [20–22 strongly inhibited the growth of the A-549 cell line, with compound 21 (GI50 = 4.80 µM) being the most effective growth inhibitor among the three. As for compounds 20 and 22, both were found to possess rather similar growth-inhibitory properties . The remaining labdane diterpenes were reported to moderately inhibit the growth of the A-549 cell line in the following order: 24 > 18 > 23 > 19 .In addition to the MCF-7 and HeLa cell lines, Suresh and his coworkers also measured the growth-inhibitory activities of the cytotoxic labdane diterpenes (ell line . AccordiH. ellipticum [E)-14,15,16-trinorlabda-8(17),11-dien-13-oic acid. The IC50 values for both these compounds were above 100 µM. Compound 29 (IC50 = 0.40 µM) was regarded as potent. Its cytotoxic activity was comparable to doxorubicin (IC50 = 0.10 µM) but found to be four and a half-folds stronger than that of ellipticine (IC50 = 1.79 µM). Compounds 16 (IC50 = 2.18 µM) and 30 (IC50 = 2.72 µM) were almost equally cytotoxic toward the NCI-H187 cells. Both labdane diterpenes were considered to be comparable to ellipticine (IC50 = 1.79 µM) in terms of their cytotoxicity [6) or weakly  cytotoxic. The rather weak cytotoxicity of compounds 20, 27, 28, and 31 upon comparison to compounds 6, 16, 29, and 30 implied that the presence of an α,β-unsaturated γ-lactone ring in the side chain played an essential role in facilitating the cytotoxic activity against the tested cell line. When the cytotoxicity of compounds 16, 29, and 30 was compared to that of compound 6, the greater activity of compounds 16, 29, and 30 led to the assumption that the potency of these three compounds could have resulted from the presence of a trans double bond which was conjugated to the α,β-unsaturated γ-lactone ring in their respective side chains in contrast to compound 6 in which the trans double bond was absent. The variation in the α,β-unsaturated γ-lactone rings of compounds 16, 29 and 30 could further explain the significant difference in the cytotoxicities between these labdane diterpenes. The substitution of position C-16 of the α,β-unsaturated γ-lactone ring in compound 16 with a hydroxyl group, as well as the substitution of position C-15 of the α,β-unsaturated γ-lactone ring in compound 30 with a methoxyl group, has lowered their cytotoxicities by almost six- to seven-fold compared to compound 29, in which none of its carbon atoms in its α,β-unsaturated γ-lactone ring were substituted with an oxygenated functional group.Songsri and Nuntawong in their investigation revealed that the NCI-H187 cell line in comparison to the MCF-7 and KB cell lines was more susceptible to the cytotoxic activity of the labdane-type diterpenes which they had isolated and characterized from the hexane and dichloromethane extracts of the rhizomes of lipticum . Among ttoxicity . The remH. gardnerianum for their cytotoxicity against the NCI-H187 cell line. Preliminary results of their REMA assay revealed that both extracts exhibited significant cytotoxicity [14, 27, 29, 32, 33, 34, and 35) were identified. The most cytotoxic labdane diterpene was compound 29 (IC50 = 0.40 µM) followed by compounds 35 (IC50 = 2.46 µM) and 34 (IC50 = 3.10 µM). Interestingly, the cytotoxicity of compound 29 was almost 4.5-fold more potent than that of ellipticine (1.79uM). The remaining compounds  only weakly inhibited the cytotoxic activity of the NCI-H187 cells [14, 27, and 32 had a trans double bond conjugated to a furan ring in their respective side chains, the presence of a hydroxyl group bonded to either position C-6 or C-7 of the decalin moieties of compounds 14 and 32 tends to have slightly increased the activity of these compounds compared to that of compound 27 in which its decalin moiety does not bear a hydroxyl group at the corresponding positions. With regards to the activity of compounds 29 and 33–35, although all four compounds have a trans double bond which is conjugated to an α,β-unsaturated γ-lactone ring in their respective side chains, the variation in the α,β-unsaturated γ-lactone rings in addition to the variation in the decalin moieties could have contributed to the differences in the extent of their cytotoxic activities. For example, the presence of a hydroxyl group at positions C-6 and C-7 in the respective decalin moieties of compounds 33 and 34 resulted in a significant decrease in their activities in contrast to compound 29 whose decalin moiety does not bear a hydroxyl group at either one of the corresponding positions. Similarly, when the activities of compounds 34 and 35 were compared to one another, although both compounds bore a hydroxyl group at position C-7 of their respective decalin moieties, the presence of a hydroxyl group bonded to the α,β-unsaturated γ-lactone ring in compound 35 slightly increased its activity compared to that of compound 34 whose α,β-unsaturated γ-lactone ring does not bear a hydroxyl group.In 2010, Kumrit et al. screened the hexane and dichloromethane extracts prepared from the rhizomes of toxicity . The ext87 cells . It was 6, isolated and characterized from the rhizomes of C. manga, exhibited a moderate cytotoxicity against the DU145 cell line, and its IC50 value was measured to be 11.21 µM [6 was once again, like in the cases of the MCF-7 and HepG2 cell lines, found to be more cytotoxic compared to the curcuminoids isolated alongside it. Demethoxycurcumin (IC50 = 21.70 µM), bisdemethoxycurcumin (IC50 = 30.10 µM), and curcumin (IC50 = 20.60 µM) were less effective in inhibiting the cytotoxic activity of the DU145 cells compared to compound 6 . The reported IC50 value of compound 6 was lower than the compound’s IC50 value against the HepG2 liver cancer cell line (IC50 = 25.33 µM), but higher than the compound’s IC50 value against the MCF-7 breast cancer cell line (IC50 = 0.59 µM), within the same study [6 is the most cytotoxic towards the MCF-7 breast cancer cells, followed by the DU145 prostate cancer cells and then finally the HepG2 liver cancer cells [DU145 is the only prostate cancer cell line reported in a study included in this review to evaluate the cytotoxicity of the labdane-type diterpenes isolated from the Zingiberaceae . Till pr11.21 µM . CompounThe results in this review were summarized from 17 studies that reported on the cytotoxicity of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes which were isolated and characterized from the Zingiberaceae against various types of cancer cell lines. This systematic review provides a comprehensive overview of the relevant studies from 2000 to 2022. This review adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).50 values for the positive control. Therefore, comparing the effectiveness of the labdane-type diterpenes in inhibiting the cytotoxic activity of a given cell line to the positive control between different studies cannot be carried out conclusively.In regards to the limitations, the studies included in this review are limited to the in vitro cytotoxic studies against the various types of cancer cell lines. Furthermore, the types of cancer cell lines included in the review are also limited and do not include every type of cancer. The several types of cancer cell lines included in this review have a very limited number of studies carried out on them. There is also a lack of consistency in the reporting of the cytotoxicities by the studies included. The cytotoxicities of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes were reported in both half-maximal inhibitory concentration and half-maximal growth inhibition. Furthermore, the studies included in this review applied different assays to measure the cytotoxic activities of the labdane-type diterpenes. The positive controls which were tested alongside the labdane-type diterpenes in the different studies were also different. Certain studies do not include the ICAs a systematic review, this review only serves to provide an overview of the existing literature and does not aim to assess the quality of the studies.Due to the various side effects of therapies currently used in the treatment of cancer, there continues to be a need for the search for alternatives. Several types of cancers, such as pancreatic, cervical, and prostate cancers, have little to no studies conducted to evaluate the cytotoxicities of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes from the Zingiberaceae. Future studies should focus on the effects of the labdane diterpenes, norlabdane diterpenes, and bislabdanic diterpenes against these cancer types that currently have fewer studies conducted, and therefore fewer data points to consider. There is also a lack of studies on the mechanism of action of the cytotoxicities against the tested cell lines. Further studies of the cytotoxicities against cancer cells should also investigate the mechanism of action in addition to the half-maximal inhibitory concentration and half-maximal growth inhibition of the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes.Alpinia, Amomum, Curcuma, Hedychium and Roscoea which exhibited cytotoxicity with IC50 < 100 µM were discussed. The subclasses of the labdane-type diterpenes which demonstrated cytotoxic activity were the labdane diterpenes, norlabdane diterpenes, and bis-labdanic diterpenes. The genus Hedychium was the richest source of active compounds.In the present review, forty-four labdane-type diterpenes from five different genera of the Zingiberaceae, namely The cytotoxicity of the labdane-type diterpenes was established on various types of cancer cell lines, in particular the most common types of cancers such as breast cancer , cervical cancer , liver cancer (HepG2 and HCC-S102 cell lines), colorectal cancer , pancreatic cancer (BxPC-3 cell line), lung cancer (A-549 and NCI-H187 cell lines), and prostate cancer (DU145 cell line).50 < 10 µM was obtained with two compounds (6 and 16) against the MCF-7 cell line, one compound (17) against the T-47D cell line, three compounds  against the MDA-MB-231 cell line, five compounds  against the KB cell line, two compounds (17 and 25) against the HeLa cell line, three compounds  against the HepG2 cell line, one compound (7) against the HCT-116 cell line, four compounds  against the HT-29 cell line, four compounds  against the A549 cell line, and five compounds  against the NCI-H187 cell line. The presence of either a γ-lactone ring or an α,β-unsaturated γ-lactone ring in the side chain of a majority of these labdane-type diterpenes could have contributed to their potencies. Among the abovementioned nineteen labdane-type diterpenes reported to have IC50 values lower than 10 µM, only compounds 16 and 17 were found to be active across several types of cell lines. Compound 16 actively inhibited the cytotoxic activities of four types of cell lines which include the MCF-7, KB, HepG,2, and NCI-H187 cell lines. Compound 17 on the other hand was effective in inhibiting the activities of the T-47D, MDA-MB-231, KB, HepG2, and HT-29 cell lines.Activity with ICThe present review thus provides an insight into the potential of the Zingiberaceae as a source of natural anticancer drugs bearing the labdane skeleton.


CDKN1A mRNA levels, decreased cell cycle-related protein expression, and increased the sub-G1 cell population in both cells. In ES2 cells, caspase-4 and GSDME were activated, whereas caspase-3 was not, indicating that bexarotene-induced cell death might be pyroptosis. A clinical setting concentration of bexarotene induced cell death through caspase-4–mediated pyroptosis in ovarian cancer cell lines. Thus, bexarotene may serve as a novel therapeutic agent for ovarian cancer.Bexarotene selectively activates retinoid X receptor, which is a commonly used anticancer agent for cutaneous T-cell lymphoma. In this study, we aimed to investigate the anticancer effect of bexarotene and its underlying mechanism in ovarian cancer in vitro. The ES2 and NIH:OVACAR3 ovarian cancer cell lines were treated with 0, 5, 10, or 20 µM of bexarotene. After 24 h, cell number measurement and lactate dehydrogenase (LDH) cytotoxicity assay were performed. The effect of bexarotene on CDKN1A expression, cell cycle-related protein, cell cycle, pyroptosis, and apoptosis was evaluated. Bexarotene reduced cell proliferation in all concentrations in both the cells. At concentrations of > 10 µM, extracellular LDH activity increased with cell rupture. Treatment using 10 µM of bexarotene increased  Although the initial clinical response is generally satisfactory, more than 70% of affected patients experience recurrences and ultimately die. Multimodality treatment with cytoreductive surgery and platinum–taxane-based chemotherapy combined with molecularly targeted drugs has shown prolonged survival. However, the overall cure rate of the disease has not considerably changed. Therefore, additional treatment strategies that can improve survival are urgently required.Ovarian cancer is the fifth most common cause of cancer-related death and the most deadly gynecological cancer in western countries2. Bexarotene is approved by the FDA and is a widely used drug for treating cutaneous manifestations in cutaneous T-cell lymphoma (CTCL)3. Moreover, this drug has gained increasing attention in cancer treatment; hence, it has been used as an off-label drug for non-small cell lung cancer4 and breast cancer5. In vitro studies have shown that bexarotene can also prevent and overcome acquired drug resistance in advanced breast cancer7, non-small cell lung cancer9, and even advanced prostate cancer10. An in vitro analysis revealed that bexarotene could kill cancer cells via apoptosis, decrease TGF-α and EGFR expressions, induce cellular senescence associated with increased p21 and p16 expressions, and promote G1 phase cell cycle arrest11. However, the precise mechanism of cell proliferation inhibition by bexarotene remains unclear.Retinoid X receptor (RXR) is a member of nuclear receptor family. RXR acts as a transcriptional factor by homodimerizing with itself or other nuclear receptor families when stimulated by their specific ligands. Bexarotene  is an RXR-selective agonist that binds and activates all three RXR isoforms  with equivalent affinity and potency13. The polymerized GSDMD N-terminal forms pores in the cell membrane to increase permeability and osmosis, thereby causing cell rupture, which is a characteristic of pyroptosis. Caspase-3, a well-known apoptotic caspase, causes pyroptosis with gasdermin E (GSDME/DFNA5). Chemotherapy drugs such as cisplatin, etoposide, and doxorubicin induce pyroptosis in tumor cells with high GSDME levels caused by caspase-314. Several anticancer drugs, such as chemotherapy drugs and miRNA, can reduce tumor viability and invasiveness by inducing tumor pyroptosis15. Hence, pyroptosis induction might be a potent anticancer drug target16.Pyroptosis is one of the regulated cell deaths triggered by certain caspases that are mainly activated by inflammation, anticancer drugs, and endoplasmic reticulum (ER) stress. Inflammatory caspases (caspase-1/4/5 in humans) cleave gasdermin D (GSDMD), which is required and sufficient for pyroptosis17. Therefore, RXR-selective retinoids, such as bexarotene, might potentially treat ovarian cancer; however, the effect of bexarotene on ovarian cancer remains unclear. Hence, this study aimed to investigate whether bexarotene could suppress the proliferation of ovarian cancer cells in vitro and better understand the antitumorigenic potential of bexarotene by exploring the precise mechanism of bexarotene on cell death, particularly pyroptosis.In epithelial ovarian cancer, retinoic acid receptors are frequently and strongly expressed and may indicate an adverse prognosis2 in room air.The ovarian clear cell carcinoma cell line (ES2) and serous ovarian carcinoma cell line (NIH:OVACAR3) were used. ES2 and NIH:OVACAR-3 cell lines were purchased from ATCC  and RIKEN BioResource Center . These ovarian cancer cell lines were routinely cultured in RPMI 1640  containing inactivated 2% fetal bovine serum  and 1% penicillin–streptomycin  at 37 °C with 5% COIn the culture medium, we seeded the cells in 12-well plates . At 80% confluence, the cells were treated with 0–20 µM of bexarotene  dissolved in dimethyl sulfoxide  for 24 h. Bexarotene is insoluble in water; hence, it was dissolved in dimethyl sulfoxide (DMSO). Therefore, all the experiments, including the untreated control, were performed with a concentration of 0.1% DMSO. The cells were then treated with trypsin–EDTA solution  and mixed with the total amount of trypan blue solution . Subsequently, the live cells were counted using a cell-counting chamber  under a stereomicroscope .Moreover, we collected the cell culture supernatant and measured the LDH activity using the LDH Cytotoxicity Assay Kit  according to the manufacturer’s protocol. The LDH levels of the cell culture supernatant derived from the bexarotene-treated cell group were normalized relative to the cultured medium derived from the control group (vehicle of the bexarotene-treated group). Each experiment was repeated at least three times, and the LDH activity was compared to that of the control group.g for 15 min at 4 °C, separated 7.5 µg of total protein in 10% or 4–15% gradient SDS-PAGE , and transferred it to a polyvinylidene fluoride (PVDF) membrane . Nonspecific binding to the PVDF membrane was blocked in Blocking one-P solution  at room temperature for 30 min. The first antibody was reacted at 4 °C for overnight. Subsequently, we washed the PVDF membrane in PBS-T and enhanced the protein signal by anti-mouse or rabbit IgG antibody  at room temperature for 1 h. We used ECL select (Roche) or ECL Prime reagent (Roche) to detect protein signals. A densitometric analysis of western blot was performed using a densitometer , and the intensity was normalized to the b-actin protein levels.We washed the cultured cells with PBS and extracted the total protein using a complete Lysis-M reagent  containing 1% Halt™ Phosphatase Inhibitor Cocktail  for 15 min at room temperature. We subsequently centrifuged the cell lysate at 15,000×CDKN1A and GAPDH  mRNA expression levels. The expression levels of measured genes were normalized relative to that of GAPDH mRNA. The 2−∆∆Cq method was used to obtain the relative quantitative value18. Supplementary Table Total RNA from cultured cells was extracted using the RNeasy Mini Kits  according to the manufacturer’s protocol. Using the SuperScript VILO cDNA Synthesis Kit , we reverse-transcribed the extracted RNA into complementary DNA (cDNA). Real-time quantitative PCR (RT-qPCR) was conducted using Light Cycler DNA Master SYBR Green I Kit  with Light Cycler Nano System (F. Hofmann–Roche Ltd). Furthermore, we evaluated the Vehicle or 10 µM–bexarotene-treated cells were cultured for 48 h and then harvested by trypsin–EDTA solution. The cells were washed twice with PBS and fixed with 70% ethanol (Fujifilm Wako) at –30 °C until cell cycle analysis. Following this, cells were incubated in 20 µg/ml RNase for 30 min after staining with 50 µg/ml propidium iodide (PI) solution (Nacharai tesque) at 4 °C for 30 min in darkness. Fluorescence intensity was analyzed using CytoFLEX flow cytometer  with CytEXpert Soft wear (BECKMAN COULTER). We analyzed 15,000 events to determine the cell cycle phase and classified cells into sub-G1, G0/G1, S, or G2/M phases according to the fluorescence intensity of the cells.Using Lipofectamine RNAiMAX , we transfected ES2 cell lines with a siRNA. The si-protein kinase R-like ER kinase  RNA, si-inositol-requiring enzyme 1-A , or a non-target siRNA (control) were used. To prepare the siRNA transfection solution for each tube, we mixed 5 pmol of siRNA with 50 μL of Opti-MEM reduced-serum medium . Concurrently, we mixed 1 μL of Lipofectamine RNAiMAX with 50 μL of Opti-MEM. Following this, the two solutions were mixed by gentle pipetting and incubated for 5 min at room temperature to allow siRNA/lipid complexes to form. The treated cells forming siRNA/lipid complexes were then incubated for 48 h at 37 °C, followed by bexarotene treatment.For detecting the target protein, we used an anti-caspase-4 antibody , anti-caspase-3 (D175) antibody , anti-GSDME antibody , anti-cyclin D1 antibody , anti-Rb antibody , anti-phospho-Rb antibody, anti-CDK4 antibody , anti-CDK6 antibody , anti-PERK antibody , and anti-β actin antibody  as the first antibodies.We used ZYVAD-FMK  dissolved in dimethyl sulfoxide  for caspase-4 inhibition experiments.t-test. All comparisons were performed using a two-sided test. In addition, P < 0.05 was considered statistically significant. All statistical data were analyzed using the JMP statistical software .The cell proliferation assay was statistically analyzed using an independent First, we examined the effect of bexarotene treatment on cell viability, cell membrane damage, and cell morphological shape to determine whether bexarotene had anticancer potential against ovarian cancer cell lines. After 24 h of bexarotene treatment, cell viability was significantly reduced dose-dependently in both the ES2 and NIH:OVACAR3 cells Fig. a, and th19. The levels of released LDH significantly increased in the 10 and 20 µM–bexarotene-treated group compared with the control group in ES2 cells . Likewise, the LDH levels in NIH:OVACAR3 cells also significantly increased in the 10 and 20 µM–bexarotene-treated group (P < 0.05). These results indicated that bexarotene induced cell death by causing plasma membrane damage in ovarian cancer cell lines.The effect of bexarotene on plasma membrane damage was evaluated by measuring the released LDH activity in the culture supernatant. In living cells, LDH is present in the cytoplasm, but it is released extracellularly due to cell membrane changes that occur during the cell death processes, such as in necroptosis and pyroptosis20. Therefore, we evaluated whether bexarotene reduced the cell viability by acting as an RXR agonist in ovarian cancer cell lines.Tanaka et al. reported that a ligand-activated RXR homodimer upregulates cyclin-dependent kinase inhibitor p21 (CDKN1A) expression by directly binding to its promoter region and regulating cell proliferationRXR-α, RXR-β, and RXR-γ in ES2 and NIH:OVACAR-3 cells using RT-PCR to confirm the RXR expression. The RXR-α and RXR-β mRNA were expressed, but RXR-γ mRNA was not in both cells (data not shown). Then, to determine whether bexarotene acted as an RXR agonist in ovarian cancer cell lines, we measured the CDKN1A mRNA expression levels after 24 h of bexarotene treatment. In both the cell lines, the administration of 10 µM bexarotene increased CDKN1A mRNA expression by approximately twofold . Meanwhile, pretreatment with ZYVAD-FMK suppressed the increase in extracellular LDH levels induced by bexarotene . These results indicate that bexarotene has two functions on ovarian cancer cells: cell proliferation suppression and caspase-4 GSDME–related pyroptosis, and these are independent actions.We then evaluated whether 10 µM–ZYVAD-FMK (caspase-4 inhibitor) pretreatment can affect pyroptosis. ZYVAD-FMK reduces the cleaved GSDME levels at 4 h after the bexarotene treatment Fig. d, S2B. T25. Thus, we examined whether bexarotene-induced ER stress in ES2 cells. We also evaluated whether bexarotene activated the unfolded protein response (UPR) signaling pathways, which are common ER stress markers, in ES2 cells. In this study, we used PERK phosphorylation, and X-binding protein 1 (XBP1) mRNA splicing as ER stress response markers.The ER stress might cause pyroptotic cell death in several cells in vitroXBP1 mRNA was spliced in an increased time-dependent manner  bexarotene-induced cell death might be caused via pyroptosis mediated by caspase-4 activation and GSDME cleavage; (2) bexarotene suppresses cell proliferation through sub-G1 cell cycle arrest; and (3) bexarotene rapidly induced ER stress but might not be related to pyroptosis.max: 10.4 μM, Targretin capsules 75 mg, interview form, Minophagen Pharmaceutical Co., Ltd., Zama, Japan). Conversely, the all-trans retinoic acid (ATRA) concentration, which is a well-investigated retinoic acid against ovarian cancer in vitro, has supraphysiologic concentrations. The ATRA concentration (sometimes as high as 10 μM) in in vitro studies was 5–50 times higher than that in the clinical setting . ATRA increases apoptosis and decreases cell proliferation, thereby proving effective against several ovarian cancer cell lines29. In the study by Lokman et al.29, the cell survival of two of five serous ovarian cancer cell lines (OVCAR-3 and OV-90) was inhibited by more than 20%; however, proliferation inhibition was weak or almost nonexistent for the other three cells . In addition, several ovarian cell lines, such as COV362, COV318, SKOV-3, and CP70, are resistant to ATRA. Contrary to ATRA, bexarotene had the potential for clinical application in ovarian cancer.Our study confirmed that bexarotene (5–20 μM) induced cell death and inhibited cell proliferation in ovarian cancer cell lines. This experimental concentration of bexarotene almost has the same or lower concentrations than those of the clinical concentration . No changes were observed in the caspases, except for caspase-4. Therefore, the other caspases were not involved in the mechanism of pyroptosis induced by bexarotene. As far as we know, this is the first report of bexarotene-induced death of pyroptotic cells in an ovarian cancer cell line. According to a research by Zhang et al., bexarotene induces apoptosis in CTCL cellsCDKN1A genes and upregulates the CDKN1A mRNA expression20. In this study, bexarotene upregulated CDKN1A, which codes a cyclin-dependent kinase inhibitor p21, and downregulated cell cycle-related proteins downstream of p21.Therefore, bexarotene also acts as an RXR agonist in ovarian cancer cells. However, several studies indicated that the accumulation of p21 could induce growth inhibition and cell cycle arrest but could not induce cell death34. Thus, the induction of CDKN1A alone could not explain bexarotene-induced cell death. However, in this study, bexarotene could induce cell death with characteristic morphological changes of pyroptosis through caspase-4 and GSDME. In addition, the knockdown of RXRα or RXRβ by siRNA could not attenuate the LDH release induced by bexarotene (Supplementary Figure The anticancer effect of bexarotene was thought to be due to its genomic function as an RXR agonist. The ligand-activated RXR homodimer binds two RXR consensus domains within the promoter region of 35. Several studies have reported that ER stress can lead to pyroptosis36. Palmitic acid and the ER stressor tunicamycin induce ER stress, leading to pyroptosis. An ER stress inhibitor can inhibit pyroptosis caused by palmitic acid or tunicamycin-induced ER stress. In this study, we have also demonstrated that the attenuation of ER stress by si-PERK and si-ERE1a could not restore bexarotene-induced pyroptosis, suggesting that bexarotene-induced pyroptosis is not mediated by ER stress in ovarian cancer cells. Hence, future research should investigate how bexarotene induces pyroptosis in ovarian cancer cells.In this study, we also showed that the bexarotene-activated ER stress in ovarian cancer cells was consistent with the findings of a previous report wherein ER stress was induced in the human neuroblastoma cell line by a high concentration of bexaroteneOur study showed that the clinical concentration of bexarotene reduces cell proliferation by inducing p21, which is still widely known and can also cause cell death, which is possibly pyroptosis, in ovarian cancer cell lines. Therefore, bexarotene is a potential anticancer drug for ovarian cancer. This study, however, was unable to elucidate the mechanisms of bexarotene-induced pyroptosis. Therefore, to better understand the pharmacological action of bexarotene, future research should further investigate the mechanisms of bexarotene-induced pyroptosis in ovarian cancer cells.Supplementary Information.


This study examines cortical organoids generated from a panel of isogenic trisomic and disomic iPSC lines (subclones) as a model of early fetal brain development in Down syndrome (DS). An initial experiment comparing organoids from one trisomic and one disomic line showed many genome-wide transcriptomic differences and modest differences in cell-type proportions, suggesting there may be a neurodevelopmental phenotype that is due to trisomy of chr21. To better control for multiple sources of variation, we undertook a highly robust study of ∼1,200 organoids using an expanded panel of six all-isogenic lines, three disomic, and three trisomic. The power of this experimental design was indicated by strong detection of the ∼1.5-fold difference in chr21 genes. However, the numerous expression differences in non-chr21 genes seen in the smaller experiment fell away, and the differences in cell-type representation between lines did not correlate with trisomy 21. Results suggest that the initial smaller experiment picked up differences between small organoid samples and individual isogenic lines, which “averaged out” in the larger panel of isogenic lines. Our results indicate that even when organoid and batch variability are better controlled for, variation between isogenic cell lines (even subclones) may obscure, or be conflated with, subtle neurodevelopmental phenotypes that may be present in ∼2nd trimester DS brain development. Interestingly, despite this variability between organoid batches and lines, and the “fetal stage” of these organoids, an increase in secreted Aβ40 peptide levels—an Alzheimer-related cellular phenotype—was more strongly associated with trisomy 21 status than were neurodevelopmental shifts in cell-type composition. Cognitive disability is a universal feature of Down Syndrome (DS), and while the genetic basis of DS is clear—trisomy 21—how an extra copy of this tiny chromosome carrying ∼250 coding genes causes this phenotype is not well understood. Nor is it clear which neural cell-types, tissue and brain regions are impacted, or when impacts occur. Many studies have raised important hypotheses for specific cell phenotypes and mechanisms, but these various findings have not been consistently supported, and in some cases are conflicting. For example, several studies have suggested that interneuron number may be decreased in DS patients and human cell models , in contIn recent years, with the advent of high-throughput sequencing approaches, studies have begun to examine differences in the transcriptomes of DS versus euploid samples, but at this early stage there are few consistent conclusions. Studies almost invariably agree that many chr21 genes are upregulated in DS tissues and cells, although the number and identity of these genes varies between studies. For example, one meta-analysis of 45 transcriptome studies found 77 chr21 genes to be consistently upregulated in DS samples  and a moMost recently, several studies have reported that trisomy 21 causes broad transcriptome-wide changes, with some studies reporting global genomic dysregulation  or the pDYRK1A, RCAN1, OLIG2, and OLIG1, DSCAM, SOD1, PCNT and several IFN-receptor genes) are hypothesized to be central to DS, although consensus has not been reached. Modestly smaller fetal brain sizes have been reported in DS fetuses; however, DS infants have developmental milestones closer to normal at birth and adults often score as more severely impacted than children. Hence, it is important to better understand when in human pre-natal and/or post-natal periods the cognitive deficits arise. This is key to therapeutic strategies, and has been studied primarily in DS mouse models . Some individuals with DS develop amyloid plaques as early as adolescence, and ∼80% show clinical dementia by ∼60–65 years , we identified many subclones that were not transgenic for XIST (but had the tet-puromycin selection gene). Some subclones were shown to be euploid by spontaneous loss of one chromosome 21, with chr21 transcriptome levels equivalent to non-isogenic normal control cells (APP) before each series of differentiations, and trisomy 21 status confirmed by RNA sequencing transcriptomics.The isogenic cell lines used here were generated and characterized as described in our prior study , and expol cells . Severalol cells . iPSCs wAs described in the supplement, we first briefly compared three organoid generation protocols  legend, Forebrain organoids were fixed for 30 min in PFA at room temperature, washed three times with PBS, and cryopreserved in 30% sucrose/PBS at 4°C overnight. Fixed organoids were embedded in O.C.T. compound , frozen in an isopropanol/dry ice slurry, and sectioned at 14 μm on a cryotome. Sections were attached to Superfrost Plus slides  and stored at −20°C until staining. Prior to staining, sections were rehydrated in PBS for 5 min, and detergent extracted in 0.5% Triton X-100  for 3 min.Immunofluorescence was performed as previously described . FixatioWhole organoids were washed once with 1X PBS and placed into 2 ml microcentrifuge tubes containing one 5 mm steel bead  and 1 ml of Trizol reagent . These samples were homogenized using the TissueLyser II instrument  on the P1 setting. Beads were then removed using a magnet and samples were either stored at −80°C or RNA extraction, DNAse treatment, and RNA cleanup was performed immediately.RQN > 7.5 and strand-specific sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina in conjunction with the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Multiplex Oligos for Illumina .RNA was extracted using TRIzol reagent  according to manufacturer’s instructions. RNA samples were cleared of contaminating genomic DNA by DNAse I  treatment for 1 h at 37°C. RNA cleanup and DNAse I removal was performed using RNeasy MinElute columns  according to manufacturer’s instructions. Clean RNA was assessed for quality on an Advanced Analytical Fragment Analyzer. All samples had an Sequencing was performed by the UMass Chan Medical School Deep Sequencing Core Facility on the Illumina HiSeq 4000 platform to a depth of ∼8 million reads/sample (2 × 50bp paired-end) in the case of the large organoid experiment or on the NextSeq 500 instrument to a depth of ∼30 million reads/sample (2 × 38bp paired-end) in the case of the pilot experiment.Reads were aligned to the GRCh37/h19 human genome build using hisat2  (v2.0.5)p-values.  Therefore, to better isolate the evidence for differential expression of genes on or off chr21—a distinction that is a priori relevant for DS studies—FDRs are computed separately for chr21 and for non-chr21 genes, denoted by FDRchr21 and FDRnon21, respectively. Since many studies do not calculate FDR for chr21 and non-chr21 separately, we also report the usual genome-wide FDR in supplemental tables and illustrate the p-value cut-off for FDR < 0.1, along with FDRchr21 < 0.1 and FDRnon21 < 0.1, on the volcano plots in p-value cut-offs are the largest p-values for which the corresponding FDR < 0.1; when none of the N off-chr21 genes satisfied FDRnon21 < 0.1 the p-value cut-off was indicated as 0.1/N, the Bonferroni cut-off—this is more stringent than the FDR cut-off in general, but if a hypothetical single gene were to satisfy the FDR cut-off it would also satisfy the Bonferroni cut-off).The false-discovery rate (FDR)  was usedhttps://data.mendeley.com/datasets/3wrtkk4w5v/2. The Birey dataset uses the Pasca protocol, which corresponds more closely to the protocol used here than the modified Lancaster protocol used for the Quadrato dataset, but this had fewer than 10 cells assigned to several of the reference cell-types. Because of this, the average of the Birey and Quadrato basis vectors was used for deconvolution in the main results reported here. Marker genes for each of the 24 clusters from the Tanaka study were downloaded from https://cells.ucsc.edu/?ds=organoidatlas and ranked by p-value with ties broken by fold-change. For cell-types corresponding to single clusters, the top 30 marker genes were used for the basis vectors. For cell-types comprising more than one cluster, the top five marker genes from each cluster were selected and the remainder of the 30 genes were selected based on rank in combined marker lists for these clusters. Ribosomal and mitochondrial genes (bsecsc function getMITRIB) (Cell-type deconvolution was performed using the DWLS-WLS (dampened weighted least-squares) method in the R package DLWS  based ontMITRIB)  were excFor tests of differential expression with cell-type representation adjustment, the estimated proportion of cortical neurons in the summed replicates was included as an additive covariate in the edgeR statistical model. .t-tests were used—these do not assume equal variance between groups so can better accommodate the reduction in variance that may happen for proportions near zero. In the large organoid study, estimated proportions were based on the summed replicates for each line, for a 3 vs. 3 comparison. Bonferroni correction was used to control for testing for differences in multiple cell-types.For tests of differences in cell-type proportions associated with trisomy 21, Welch’s The R package ggplot2  was used48-h old media was removed from tissue culture wells containing individual organoids. Media was immediately placed on ice and centrifuged at 2,000 rcf for 5 min to remove cell debris. Media supernatant was stored at −80°C. ELISA was performed using the ultrasensitive Amyloid beta ELISA kit from Invitrogen   per manuThe R package drm  was usedP-values and confidence intervals were computed with the contest function in the R package lmerTest (p = 0.71), and similar results were obtained by averaging un-normalized log2 Aβ concentrations for each cell-line and performing a t-test on these 3 vs. 3 averages. This use of RNA levels as a proxy for organoid size/cell-count improves reliability of the comparisons in what are nonetheless approximate Aβ levels in this analysis.Differences in Aβ concentration will be impacted by organoid cell-counts, which we could not directly measure. So, we used RNA concentration as a proxy for cell-count and included it as an additive covariate in a linear mixed-effect model along with fixed effects for trisomy state and batch and random effects for cell line and its interaction with batch [lmer function in the R package lme4 ]. P-valulmerTest  using SaThe results presented detail the progression of experimental design improvements based on results of our initial observations. The last and largest experiment was formulated from lessons learned from our initial cerebral organoid studies, which highlighted the need to address several sources of variation that are often present in iPSC modeling, but not due to trisomy 21. DS studies have reported numerous phenotypic and transcriptional differences attributed to trisomy in human or mouse neural tissues and cells; however, it is often difficult to know whether other potential differences between samples have been ruled out. In our initial experiments, comparing individual isogenic organoids from one trisomic and one disomic line, we found intriguing differences. Given this, we then sought to determine whether these differences might be accounted for by variation (unrelated to trisomy 21) between organoids, experiments, or cell lines—even isogenic cell lines. Realizing the need to expand the experimental design led us to focus on quantitative transcriptome analyses comparing hundreds of organoids from experimental repetitions, and, importantly, generated from additional all-isogenic iPSC lines. While the need for large numbers of organoids to control for variation between organoids was not unanticipated, our results indicate that the number of isogenic iPSC lines needed to confidently investigate a potentially subtle neurodevelopmental phenotype was more than initially expected.Several 3D cell culture models of cerebral development have recently been developed, each with its own set of advantages and drawbacks. We first created organoids using three different protocols  to identWe had initially planned to examine differences between organoids using cytological staining for specific cell-type markers. For example, prolonged culture with this protocol generates significant numbers of GFAP-expressing cells, which include astrocytes  . The figOur first pilot RNA sequencing experiment used bulk RNA-seq of 10 organoids aged for 160 days, five from a trisomic  line and five from an isogenic euploid control line. Isogenic lines have the same genetic background, and comparison of subclones derived from the same iPS parental line avoids differences in reprograming or somatic cell of origin. The overall strategy was to generate bulk sequence data and use published gene sets and expression profiles for different cell-types to deconvolve the cell-type representation in each sample. In this first experiment we chose to evaluate the variation between individual organoids by sequencing the 10 organoids individually.FDR < 0.1 . As explained in the methods, to avoid diluting the signal for differential expression among the small proportion of genes on chr21, we also computed FDRs separately for chr21 genes (designated FDRchr21) and non-chr21 genes (FDRnon21). Here because there were so many DEGs off of chr21 this had only a modest effect, with 111 chr21 genes having FDRchr21 < 0.1 and 5,626 non-chr21 genes having FDRnon21 < 0.1. These 111 chr21 genes included 10 with higher expression in disomic than trisomic samples, which may reflect differences between particular cell-lines or organoid-batches. The DEGs include genes with various non-coding biotypes and genes with lowish average expression, which contribute to the high number of non-chr21 DEGs, but 78%  were for protein-coding genes, 87%  for genes with average CPM > 1, and 75%  for both. As discussed in the introduction, differences of several hundred up to a few thousand non-chromosome 21 genes are a common finding in published studies of DS.We generated RNA-seq data for each of the 10 organoids to a depth of ∼30 million reads and then compared RNA-seq data for the five trisomic and five disomic organoids. This data showed a clear difference in overall expression of genes from chr21 between individual organoids from the trisomic versus the disomic iPSC lines . In testIn p < 0.05 by Welch’s t-test before multiple hypotheses correction) for three cell-types—glial progenitor cells, cortical neurons, and interneuron—but after Bonferroni-correction these were no longer statistically significant, with p ≥ 0.23 for all cell-types   . HoweverEven if consistent significant differences had been seen between organoids generated from these two cell isogenic cell lines, a key question would remain as to whether that difference is due to the presence of trisomy 21, or to other sources of variation between samples, including between isogenic cell lines cultured separately . Since hThe above findings provide suggestive differences in organoid development from the two cell lines, although these were only nominally significant. A more powerful experimental design could affirm or discount that these differences are consistently correlated with trisomy 21. The above results indicated that many more organoids should be examined for each line, but we also recognized that differences observed between any two particular lines could reflect differences unrelated to trisomy 21. Thus, the experimental design was greatly expanded to include many more organoids, more isogenic lines, and more experimental repetitions , in ordeInitial sequencing analysis confirmed the expected ∼1.5 fold higher overall chromosome 21 expression in all trisomic versus all disomic lines .We next used the bulk transcriptome data to estimate the cell-type composition, as in the pilot study. The most highly represented cell-types were cortical neurons and interneurons, which form neighboring clusters in the UMAP projection in the study from which these cell-types were defined . There wp-values > 0.2 before multiple hypothesis correction and all Bonferroni-corrected p-values equal to 1 . Even with several hundred organoids compared in four replicate experiments, the statistical power is limited by the number of isogenic trisomic and disomic lines. Multiple organoids from the same “batch” or replicate batches (experiments) from the same cell line are not independent samples for comparing the effects of trisomy 21, because results will be impacted by the particular size propensity of particular cell lines (or batches) compared.Irrespective of the cell-type composition, we also considered whether differences in organoid size may be correlated with trisomy 21. The presence of the extra chromosome may confer increased cell stress  or cell We emphasize that these findings do not discount that there could be a mild growth disadvantage conferred by trisomy 21, even potentially in organoids, but results here highlight that the inter-cell-line (and inter-batch) variability can be a complicating factor in such studies. In fact, we found decreased proliferation of undifferentiated trisomic iPS cells in a prior study that directly compared cultures of the same cell line with and without induced silencing of one chr21 . Comparip-values that reflect real biological differences between lines or samples, but, importantly, these are not necessarily due to trisomy 21. This is a form of pseudoreplication  of all analyzed chr21 genes. Most chr21 DEGs were at or near the theoretical 1.5-fold higher expression expected in trisomic cells . Neither GFAP nor the DLX genes noted in the smaller pilot study were near the cut-off for significance in the expanded study  of genes expressed at >1 CPM differentially expressed at ic cells ; this ised study .per se. Given the paucity of non-chr21 DEGs in this expanded study, we also considered that inconsistent cell-type representations between the various cell lines/samples will increase expression variability that could weaken detection of gene expression changes between trisomy and disomy groups. To address this, we tested differential expression using a model that accounts for the estimated proportion of cortical neurons in each sample, as this was the cell-type with highest variability between samples (based on variance and IQR). As seen in FDRchr21 < 0.1 from 139 to 143, and genes off of chr21 with FDRnon21 < 0.1 remaining at zero (and just one at FDR < 0.1). Results from tests of differential expression without and with adjustment for cortical neuron proportions are provided in This indicates that the extensive genome-wide differences found in the pilot study  reflect The strongest conclusion from these results is that the presence of widespread differences in expression in the one-line vs. one-line comparison is not validated with a stronger experimental design. Other biological variation, such as in cell-type representation, may be associated with hundreds or more non-chr21 DEGs, not necessarily a direct or indirect effect of trisomy 21. While this overall finding is clear, whether any individual gene is identified as a DEG can be modestly impacted by even small differences in estimated cell-type proportions and specific cut-offs. Thus, changes in the deconvolution methodology, filtering of lowly expressed genes and other computational details can affect whether a few off-chr21 genes are identified as significant, as in our earlier analysis of this data . Hence, Aβ to contrast detection of this neurodegenerative pathology to the neurodevelopmental results. We isolated media from each organoid pool and analyzed whether an increase in secreted Aβ would be detected in these fetal stage organoids, using ELISA for Aβ40 (see Methods). p = 0.068; see Methods) than differences related to neurodevelopment .While an in-depth study of AD-related cell phenotypes is the subject of a separate study, we include here a limited analysis of in utero imaging in large numbers of developing fetuses.This study began with the expectation that isogenic comparisons of trisomic and disomic cortical organoids, as models of early fetal neurodevelopment, would reveal differences in cell-types and pathways caused by trisomy 21. Understanding how and when brain development and/or function is impacted in DS is critical to understanding the biology of trisomy 21 and to assess therapeutic prospects to mitigate cognitive or neurological deficits in DS . Given tNumerous studies report a variety of differences comparing trisomic and euploid samples, including in various mouse models, different human DS tissues, and various cell-based models. Inconsistencies in findings between studies may be due to differences in the systems examined, or in some cases limitations in sample size, etc. Human iPSC studies have the potential to provide more controlled comparisons between trisomic and euploid cells/organoids in comparable developmental/functional states. However, iPSC cultures are prone to environmental changes that can affect experimental results  and orgaFrom the start we used a totally isogenic system, using isolated lines (subclones) derived from a single reprograming event, avoiding the differences between isogenic lines from distinct reprograming events or cell of origin effects, which can manifest as differences in neural differentiation potential . BecauseFDRchr21 < 0.1). This was more than the 58% of chr21 genes satisfying the same criteria in the smaller (pilot) experiment, and more chr21 DEGs than reported in many studies of DS tissues or cells, which, paradoxically—and as in our pilot study—report many more off-chr21 DEGs than found in the expanded experiment here  for each organoid experiment  repository, accession number JC and JL conceived and designed the study. JC performed the experiments. JC, OK, and JL analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.