Part 2

Hypoxic challenge triggers RIP-dependent necroptosis in human colorectal carcinoma cells

Human colorectal carcinoma Caco-2 cells were exposed to normoxia (Nx) or hypoxia

(Hx) in glucose-free media (Φ) for various time points, and a time-dependent increase

of LDH leakage was observed in Hx+Φ but not Nx+Φ cells (Figure 12A). Live images

revealed cytosolic vacuolation, widening intercellular space, and cell detachment in a

timely order following hypoxic challenge, whereas no morphological change was

observed in normoxic counterparts (Figure 12B). No sign of apoptosis was found after

hypoxic challenge as evidenced by the lack of oligonucleosome formation and

caspase-3 activation (Figure 13). Similar results of hypoxia-induced cell necrosis were

seen in another human colorectal tumor cell line HT29 (Figure 14A and B).

The mitochondrial transmembrane potential was determined by using a cationic

JC-1 dye. Exposure to hypoxia resulted in a transient increase and then decline in red

fluorescence intensity (the aggregated form of JC-1) followed by a display of green

fluorescence (the monomer form of JC-1) in the cytoplasm at later time points (Figure

12C). Quantification results indicated that the ratios of J-aggregate/monomer in cells

after 8- and 24-hr hypoxia were 221.1 ± 49.0% and 20.5 ± 2.8%, respectively, of that of

the normoxic controls (Figure 12D), suggesting that hypoxia caused a transient

hyperpolarization and a final collapse of mitochondrial transmembrane potential.

Furthermore, plasma membrane disintegration paralleled with loss of tight

junctions in hypoxic cells, evidenced by reduction of transepithelial electrical resistance

(TER), increase of apical-to-basolateral dextran flux, and structural disruption of zonula

occluden-1 (ZO-1) (Figure 12E-G).

Pretreatment with necrostatin-1 (a specific RIP1 inhibitor) and gene silencing of

RIP1 reduced the level of LDH leakage caused by hypoxic challenge (Figure 15A and

3-2B). A ~50% knock down of RIP1 protein by siRNA was confirmed by Western blots

(Figure 15B). Using immunoprecipitation and 32P kinase assays, formation of RIP1/3

complex and phosphorylation of RIP1 were found in Hx+Φ but not Nx+Φ cells (Figure

16A), indicating the activation of RIP1/3 signaling. The hypoxia-induced morphological

damage and cell detachment were also inhibited by necrostatin-1 (Figure 15C).

However, the mitochondrial transmembrane potential change was not reverted by

necrostatin-1 (Figure 15D), suggesting that RIP1 activation may not be upstream of

mitochondrial dysfunction. In hypoxic cells treated with necrostain-1, a transient

increase in red fluorescence was seen after 8 hrs followed by a switch to green

fluorescence after 24 hrs (Figure 15D), of which the quantification results of JC-1

staining were 277.2 ± 25.2% and 40.2 ± 13.6%, compared to normoxic controls with

necrostain-1 at respective time points.

Glucose uptake abolishes hypoxia-induced RIP signaling and necroptosis

Administration of glucose (25 mM) reduced the RIP1/3 complex formation and

phosphorylation (Figure 16A) and decreased LDH leakage in hypoxic cells in a

dose-dependent manner (Figure 16B). Non-metabolizable sugar analogs (i.e. 3-OMG

and mannitol) or glutamate did not reduce the LDH activity (Figure 17). Moreover,

glucose addition also ablated hypoxia-induced morphological damages (data not shown),

mitochondrial transmembrane potential damage (Figure 16C), and tight junctional

disruption (Figure 16D-F). Addition of glucose did not modify the apoptotic levels in

hypoxic cells (data not shown).

To confirm that cells still perceive hypoxic stress after glucose addition, activation

of HIF1α and hypoxia-targeted genes were examined. Nuclear translocation of HIF1α

(Figure 18A) correlated with increased expression of GLUT-1 and -4 at the

transcriptional and translational levels in hypoxic cells given glucose (Figure 18B-D).

The experiment was not carried out on hypoxic cells in glucose-free media due to cell

necrosis (i.e. plasma membrane disintegration and release of cellular contents).

Moreover, similar results of glucose-mediated death resistance, HIF1α activation, and

GLUT upregulation were also seen in HT29 cells under hypoxic stress (Figure. 19A, C,

D).

Anaerobic glycolytic metabolism is involved in anti-necrotic resistance to hypoxia stress

To verify the metabolic process that is involved in death resistance, cells were pretreated

with iodoacetate (IA, a glycolytic inhibitor to GPD) and UK5399 (UK, a MPC inhibitor)

prior to hypoxic challenge in the presence of glucose. Blockade of glucose-mediated

resistance was noted in cells pretreated with IA, whereas no inhibitory effect was seen

with UK (Figure 20A), suggesting that glycolytic products unrelated to TCA cycles

were involved in anti-necrotic mechanisms. For hypoxic cells in glucose-free media, IA

and UK had no effect on LDH activity (data not shown).

The intracellular ATP, pyruvate, lactate contents were next quantified to examine

the bioenergetic status of cells. A significant reduction of intracellular ATP and pyruvate

levels were seen in hypoxic cells compared to their normoxic controls in glucose-free

media (Figure 20B and C), whereas comparable lactate production was noted between

the two groups (Figure 20D). Addition of glucose partially prevented the drop of ATP

and pyruvate caused by hypoxia, and significantly increased the lactate contents (Figure

20B-D). The effect of IA was confirmed by lower levels of ATP, pyruvate, and lactate

production compared to those given glucose without inhibitors in both normoxic and

hypoxic conditions (Figure 20B-D). In contrast, UK had no effect on these parameters

(Figure 20B-D).

Pyruvate is involved in death resistance through mitochondrial superoxide scavenging without restoration of cellular energy

The specific role of pyruvate in the mechanisms of death resistance was examined by

replacing glucose with cell-permeable ethyl pyruvate derivative in hypoxic cells.

Addition of pyruvate derivative significantly reduced the LDH leakage, RIP1/3 complex

formation, and morphological damage in hypoxic cells (Figure 21A-C). However, the

ATP levels in cells given pyruvate were comparable to those without supplementation

(Figure 21D). Presence of pyruvate neither altered cellular ATP contents nor suppressed

dextran flux in hypoxic cells (Figure 21E), indicating that death resistance by pyruvate

was uncoupled with ATP production and energy-dependent processes (e.g. tight

junctional restoration). Unlike glucose, pyruvate did not suppress hypoxia-induced

mitochondrial transmembrane potential changes (Figure 21F). These results suggest that

pyruvate confers resistance to necroptosis through an alternative, energy-independent

mechanism.

Generation of mitochondria-derived oxidative free radicals has been implicated in

the cell necrotic pathways triggered by cytotoxic agents (Declercq et al., 2009; Zhang et

al., 2009), and we sought to examine its role in hypoxic necrosis. Increased mean

fluorescence of MitoSox (an indicator of mitochondrial superoxide production) was

observed in hypoxic cells compared to normoxic controls in glucose-free media (Figure

22A). Decreasing the mitochondrial superoxide levels with butylated hydroxyanisole

(BHA, a free radical scavenger) and apocynin (an inhibitor to nicotinamide adenine

dinucleotide phosphate (NADPH) oxidase) (Figure 22A) led to partial inhibition of

LDH leakage in hypoxic cells (Figure 22B). Moreover, pretreatment with BHA also

diminished the RIP1/3 complex formation (Figure 22C). These results indicate that ROS

production which is upstream of RIP signaling is involved in hypoxia-induced

necroptotic pathways.

The modulatory effect of glucose and pyruvate on free radical levels was next

addressed. Addition of glucose decreased hypoxia-induced mitochondrial ROS levels

(Figure 22D), but did not alter the redox activities of catalase, superoxide dismutase,

glutathione reductase, or glutathione-S-transferase (Figure 23), suggesting a

non-enzymatic scavenging mechanism. The glucose-mediated reduction of

mitochondrial ROS may be inhibited by pretreatment with IA but not UK (Figure 22D).

Lastly, replacing glucose with cell-permeable pyruvate derivative also significantly

suppressed the mitochondrial ROS levels in hypoxic cells (Figure 22D). Similar results

of pyruvate-mediated resistance were seen in HT29 cells under hypoxic stress (Figure

19B, E).